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The action of picrotoxin and bicuculline on rat caudate neurons inhibited by GABA
Brain Research, 102 (1976) 379-384
379
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
The action of picrotoxin and bicucuUine on rat caudate neurons inhibited by GABA
G. BERNARDI, M. G. MARCIANI, C. MOROCUTTI AND P. GIACOMINI 11. Clinica delle Malattie Nervose e Mentali, Universitdt di Roma, 00185 Rome (Italy)
(Accepted October 8th, 1975)
Since picrotoxin and bicuculline have been considered as selective antagonists of the inhibitory 7,-aminobutyric acid (GABA) in vertebrate nervous system2-4,6,10,11, these alkaloids have been extensively used to examine the sites where GABA may act as an inhibitory synaptic transmitter. However, several authors have questioned their value as antagonists of GABA in the mammalian central nervous systemV,S,l~, 14. Therefore experiments were performed to determine the action of these alkaloids on some interneuronal systems of rat caudate in which GABA mimicked the inhibitory transmitter 1. Male Wistar rats (250-300 g) were anesthetized with ether, immobilized with succinylcholine and artificially ventilated. Body temperature was kept constant by means of a heating pad. Frontal cortex and globus pallidus (AP 1 ; L 3 ; H 7) 5 were stimulated with bipolar electrodes. Intracellular recordings from the head of the caudate nucleus (AP --1.5; L 2-4; H 3-6) were obtained with a single micropipette glued in parallel with the shanks of a 3-barrelled pipette used for application of drugs. The recording pipette was filled with potassium citrate (1.6 M) and protruded 50-100 # m beyond the tip of the 3-barrelled pipette. One barrel contained GABA (1 M, pH 3), the second barrel picrotoxin (4.5 m M in 165 m M NaCl, pH 7) and the third one bicuculline (5 m M in 165 m M NaC1, pH 3). Picrotoxin and bicuculline were also injected intravenously. The membrane resistance of neurons (2 cells) was calculated from the amplitude of an injected pulse (25 msec, hyperpolarizing current). Positions of the stimulating and recording electrodes were controlled histologically. Field potentials were recorded in the caudate nucleus contralaterally to the one where the micropipette was inserted. E K G was routinally monitored. The results were obtained from intracellular recordings of 18 neurons of rat caudate nucleus whose electrical activity was inhibited by GABA iontophoretically applied (10-100 nA). Cortical and globus pallidus stimulation always evoked EPSP1PSP sequences o f long duration (150-300 msec). Picrotoxin, iontophoretically applied (80-300 nA), blocked GABA inhibition of the evoked and 'spontaneous' action potentials, but did not reduce the postsynaptic inhibition. Bicuculline (50-250 nA) had analogous effects, and in some neurons the postsynaptic inhibition was
380 A
B
_,, .V]
tT,
"
i _STraY
-3S
-~
Fig. 1. Effect o f picrotoxin (120 #g/kg i.v,) on E P S P - I P S P sequence evoked in a caudate neuron
(lower trace) and on the field potential evoked in the contralateral caudate (upper trace). Cortical stimulation (black triangle). A: control. B-E: picrotoxin effect after 22, 26, 32 and 35 sec from injection. F: seizures and cell deterioration after 42 sec. Note in B the depolarization of the membrane potential and the frequency increase of the action potentials; in C-E further depolarization occurs with decrease of IPSP amplitude and appearance of spikes during IPSP. The field potential is not modified. enhanced. However, considering that most of the axons in the rat caudate nucleus make synapses on thin dendrites 9, while the pipettes are presumed to be located close to the cell body, this distribution might account for the fact that bicuculline and picrotoxin did not block the postsynaptic inhibition. For this reason the two alkaloids were injected intravenously. Picrotoxin (50-200 #g/kg) quickly depolarized the cell membrane and decreased the IPSP amplitude while spikes appeared during the IPSP (Fig. 1B-E). The field potential recorded in the contralateral caudate nucleus did not show any change. E K G was usually constant. Subsequently seizures appeared and a further depolarization of the cell membrane potential followed (Fig. IF). Bicuculline (20-100/~g/kg) produced slow changes of the membrane potential, depolarizing some neurons (Fig. 2) and hyperpolarizing others (Fig. 3E:-H). Independently from the membrane potential variations, the IPSP amplitude decreased
381 A I
EBG
BI
_44
~4$
-49mV
SOO p v I
_44mV
_4~
I'Immm
Fig. 2. Effect of bicuculline (70/~g/kg i.v.) on EPSP-IPSP sequence (lower trace) and on the evoked potential (upper trace). Cortical stimulation (black triangle). A: control. B-E: effect of bicuculline after 19, 30, 33 and 37 sec from injection. F: appearance of seizures after 45 sec. In B note the membrane potential depolarization and the flattening of IPSP. In C-E note the appearance of a longlasting depolarizing wave on which spikes appear. The amplitude of the evoked field potential is increased.
till flattening (Figs. 2B and 3E). In this phase the frequency of the spikes did n o t change. Then, the stimulation evoked a depolarizing wave, increasing in amplitude and duration (up to 1 sec) (Figs. 2 C - E and 3F-I). The evoked field potential showed an increase in amplitude. E K G was not modified. Usually the seizures that followed were associated with m e m b r a n e potential depolarization (Fig. 2F). In two neurons in which m e m b r a n e resistance was measured, an increase o f resistance was observed during the bicuculline action (not illustrated). Fig. 3 shows that the different effects o f picrotoxin and bicuculline on different caudate cells are also observed when the alkaloids are applied onto the same neuron, These results eliminate the possibility that different responses are related to different kinds o f cells. The results show that picrotoxin and bicuculline act differently on rat caudate neurons inhibited by G A B A . Picrotoxin depolarizes the cell m e m b r a n e and decreases the I P S P amplitude. Spikes appear during the IPSP. The bicuculline action
382
_S,I,,tV
_SSmV
-60
_114
. . . . .
' ~ :~
-----
-I1! Fig. 3. Effects of picrotoxin (90/~g/kg i.v.) and bicuculline (50 #g/kg i.v.) on EPSP-IPSP sequence (lower trace) and on the evoked field potential (upper trace). Cortical stimulation (black triang!e), A: control. B and C: effect of picrotoxin after 30 and 57 sec from injection. The field potential is not modified. D: control 70 rain later. E - I : effect of bicuculline after 21, 30, 36, 43 and 50 sec from injection. The field potential is increased. Note in B the membrane potential depolarization and the decrease of the IPSP amplitude. In C note spike inactivation. In E - H there is hyperpolarization of the cell membrane after bicucuUine and gradual increase in amplitude and duration of the depolarizing wave. In I there is spike inactivation, while the wave maintains long duration and high amplitude although the membrane potential is more depolarized than the control.
383 is more powerful, but more complex. The slow changes of the membrane potential may be due to excitatory or inhibitory inputs coming from neurons activated by bicuculline, while the IPSP flattening, independent of the membrane potential changes, suggests a blockade of the mechanisms mediated by GABA. The long-lasting depolarizing wave could be either a reversal of the IPSP or the prolonged EPSP. However, considering that the IPSP equilibrium potential (Fig. 2B) and the depolarizing wave (Fig. 2D and E) are at the same resting potential (--44 mV), different ionic mechanisms are probably involved in these two responses. Analogous results can be observed in the neuron shown in Fig. 3E and H. Besides, the depolarizing wave persists when the membrane potential in the same neuron shifts from hyperpolarizing (Fig. 3G; - - 6 4 mV) to depolarizing (Fig. 3I; --51 mV) levels as regards the IPSP equilibrium potential (Fig. 3E; - - 6 0 mV). Thus, the evoked depolarizing wave seems to be a prolonged EPSP and not a reversed IPSP. The resistance increase, observed during bicuculline injection, could account in part for the increase in EPSP amplitude and duration. Shank et al. have already described in invertebrates the progressive increase of EPSP amplitude and duration and have related this finding to an increase of the membrane resistance due to the non-specific action of bicuculline 12. Finally we emphasize that GABA antagonism at the neuronal level, observed following the systemic injection of these alkaloids, precedes the seizures in the rat caudate nucleus, in contrast to findings by Nicoll in the olfactory bulb 1°. In conclusion, our results show that picrotoxin blocks GABA inhibition in the caudate. It depolarizes the neuron and decreases the IPSP amplitude whi)e spikes appear during the 1PSP. However, these data do not show whether the picrotoxin antagonism is specific or non-specific. The action of bicuculline is different and its effects seem to rule out the possibility that this alkaloid is a specific antagonist of GABA. Supported by C.N.R. Grant No. 74.00147.04.
l BERNARD1,G., MARCIANI,M. G., MOROCUTTI,C., AND GIACOMINI,P., The action of GABA on rat caudate neurons recorded intracellularly, Brain Research, 92 0975) 511 515. 2 CURTIS,D. R., DUGGAN,A. W., FELIX, D., AND JOHNSTON,G. A. R., GABA, bicuculline and central inhibition, Nature (Lond.), 226 (1970) 1222-1224. 3 CURTIS,D. R., DUGGAN,A. W., FELIX,D., ANDJOHNSTON,G. A. R., Bicuculline, an antagonist of GABA and synaptic inhibition in the spinal cord of the cat, Brain Research, 32 (1971) 69 96.
4 CURTIS,D. R., DUGGAN,A. W., FELIX,D., JOHNSTON,G. A. R., ANDMCLENNAN,H., Antagonism between bicuculline and GABA in the cat brain, Brain Research, 33 (1971) 57-73. 5 FIFKOVA,E., AND MARSALA, J., Stereotaxic atlas for the rat. In J. BURE~, M. PETRAN AND J. ZAClJAR (Eds.), Electrophysiological Methods in Biological Research, Academic Press, New York, 1967, p. 653. 6 GALINDO,A., GABA-picrotoxin interaction in the mammalian CNS, Brain Research, 14 (1969) 763-767. 7 GODFRAIND,J. M., KRNJEVI(~,K., AND PUMAINR., Doubtful value of bicuculline as a specific antagonist of GABA, Nature (Land.), 228 (1970) 675 676. 8 HILL,R. G., SIMMONDS,M. A., ANDSTRAUGHAN,D. W., A comparative study of some convulsant substances as GABA antagonists in the feline cerebral cortex, Brit. J. Pharmacol. 49 (1973) 37-51.
384 9 MORI, S., Some observations on the fine structure of the corpus striatum of the rat brain, Z, Zellfi~rseh., 70 (1966) 461 488. 10 NIcOLL, R. A., Pharmacological evidence for GABA as the transmitter in the granule celt inhibition in the olfactory bulb, Brain Research, 35 (1971) 137-149. 11 OSATA, K., AND HIGHSX~I~, S. M., Blocking by picrotoxin of both vestibulary inhibition and G A B A action on rabbit oculomotor neurons, Brain Research, 18 (1970) 538-541. 12 SHANK, R. P., PONG, S. F., FREEMAN, A. R., AND GRAHAM, L. T., Bicuculline and picrotoxin as antagonists of 3,-aminobutyrate and neuromuscular inhibition in the lobster, Brain Research, 72 (1974) 71-78. 13 STRAUGHAN, D. W., Convulsant drugs: amino acid antagonism and central inhibition, Neuropharmacology, 13 (1974) 495-508. 14 STRAUGHAN, D. W., NEA~,, M. J., SIMMONDS, M. A., COLLINS, G. C. S., ANO HILL, R. G., Evaluation of bicuculline as a GABA antagonist, Nature (Lond.), 223 (1971) 352 354.
379
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
The action of picrotoxin and bicucuUine on rat caudate neurons inhibited by GABA
G. BERNARDI, M. G. MARCIANI, C. MOROCUTTI AND P. GIACOMINI 11. Clinica delle Malattie Nervose e Mentali, Universitdt di Roma, 00185 Rome (Italy)
(Accepted October 8th, 1975)
Since picrotoxin and bicuculline have been considered as selective antagonists of the inhibitory 7,-aminobutyric acid (GABA) in vertebrate nervous system2-4,6,10,11, these alkaloids have been extensively used to examine the sites where GABA may act as an inhibitory synaptic transmitter. However, several authors have questioned their value as antagonists of GABA in the mammalian central nervous systemV,S,l~, 14. Therefore experiments were performed to determine the action of these alkaloids on some interneuronal systems of rat caudate in which GABA mimicked the inhibitory transmitter 1. Male Wistar rats (250-300 g) were anesthetized with ether, immobilized with succinylcholine and artificially ventilated. Body temperature was kept constant by means of a heating pad. Frontal cortex and globus pallidus (AP 1 ; L 3 ; H 7) 5 were stimulated with bipolar electrodes. Intracellular recordings from the head of the caudate nucleus (AP --1.5; L 2-4; H 3-6) were obtained with a single micropipette glued in parallel with the shanks of a 3-barrelled pipette used for application of drugs. The recording pipette was filled with potassium citrate (1.6 M) and protruded 50-100 # m beyond the tip of the 3-barrelled pipette. One barrel contained GABA (1 M, pH 3), the second barrel picrotoxin (4.5 m M in 165 m M NaCl, pH 7) and the third one bicuculline (5 m M in 165 m M NaC1, pH 3). Picrotoxin and bicuculline were also injected intravenously. The membrane resistance of neurons (2 cells) was calculated from the amplitude of an injected pulse (25 msec, hyperpolarizing current). Positions of the stimulating and recording electrodes were controlled histologically. Field potentials were recorded in the caudate nucleus contralaterally to the one where the micropipette was inserted. E K G was routinally monitored. The results were obtained from intracellular recordings of 18 neurons of rat caudate nucleus whose electrical activity was inhibited by GABA iontophoretically applied (10-100 nA). Cortical and globus pallidus stimulation always evoked EPSP1PSP sequences o f long duration (150-300 msec). Picrotoxin, iontophoretically applied (80-300 nA), blocked GABA inhibition of the evoked and 'spontaneous' action potentials, but did not reduce the postsynaptic inhibition. Bicuculline (50-250 nA) had analogous effects, and in some neurons the postsynaptic inhibition was
380 A
B
_,, .V]
tT,
"
i _STraY
-3S
-~
Fig. 1. Effect o f picrotoxin (120 #g/kg i.v,) on E P S P - I P S P sequence evoked in a caudate neuron
(lower trace) and on the field potential evoked in the contralateral caudate (upper trace). Cortical stimulation (black triangle). A: control. B-E: picrotoxin effect after 22, 26, 32 and 35 sec from injection. F: seizures and cell deterioration after 42 sec. Note in B the depolarization of the membrane potential and the frequency increase of the action potentials; in C-E further depolarization occurs with decrease of IPSP amplitude and appearance of spikes during IPSP. The field potential is not modified. enhanced. However, considering that most of the axons in the rat caudate nucleus make synapses on thin dendrites 9, while the pipettes are presumed to be located close to the cell body, this distribution might account for the fact that bicuculline and picrotoxin did not block the postsynaptic inhibition. For this reason the two alkaloids were injected intravenously. Picrotoxin (50-200 #g/kg) quickly depolarized the cell membrane and decreased the IPSP amplitude while spikes appeared during the IPSP (Fig. 1B-E). The field potential recorded in the contralateral caudate nucleus did not show any change. E K G was usually constant. Subsequently seizures appeared and a further depolarization of the cell membrane potential followed (Fig. IF). Bicuculline (20-100/~g/kg) produced slow changes of the membrane potential, depolarizing some neurons (Fig. 2) and hyperpolarizing others (Fig. 3E:-H). Independently from the membrane potential variations, the IPSP amplitude decreased
381 A I
EBG
BI
_44
~4$
-49mV
SOO p v I
_44mV
_4~
I'Immm
Fig. 2. Effect of bicuculline (70/~g/kg i.v.) on EPSP-IPSP sequence (lower trace) and on the evoked potential (upper trace). Cortical stimulation (black triangle). A: control. B-E: effect of bicuculline after 19, 30, 33 and 37 sec from injection. F: appearance of seizures after 45 sec. In B note the membrane potential depolarization and the flattening of IPSP. In C-E note the appearance of a longlasting depolarizing wave on which spikes appear. The amplitude of the evoked field potential is increased.
till flattening (Figs. 2B and 3E). In this phase the frequency of the spikes did n o t change. Then, the stimulation evoked a depolarizing wave, increasing in amplitude and duration (up to 1 sec) (Figs. 2 C - E and 3F-I). The evoked field potential showed an increase in amplitude. E K G was not modified. Usually the seizures that followed were associated with m e m b r a n e potential depolarization (Fig. 2F). In two neurons in which m e m b r a n e resistance was measured, an increase o f resistance was observed during the bicuculline action (not illustrated). Fig. 3 shows that the different effects o f picrotoxin and bicuculline on different caudate cells are also observed when the alkaloids are applied onto the same neuron, These results eliminate the possibility that different responses are related to different kinds o f cells. The results show that picrotoxin and bicuculline act differently on rat caudate neurons inhibited by G A B A . Picrotoxin depolarizes the cell m e m b r a n e and decreases the I P S P amplitude. Spikes appear during the IPSP. The bicuculline action
382
_S,I,,tV
_SSmV
-60
_114
. . . . .
' ~ :~
-----
-I1! Fig. 3. Effects of picrotoxin (90/~g/kg i.v.) and bicuculline (50 #g/kg i.v.) on EPSP-IPSP sequence (lower trace) and on the evoked field potential (upper trace). Cortical stimulation (black triang!e), A: control. B and C: effect of picrotoxin after 30 and 57 sec from injection. The field potential is not modified. D: control 70 rain later. E - I : effect of bicuculline after 21, 30, 36, 43 and 50 sec from injection. The field potential is increased. Note in B the membrane potential depolarization and the decrease of the IPSP amplitude. In C note spike inactivation. In E - H there is hyperpolarization of the cell membrane after bicucuUine and gradual increase in amplitude and duration of the depolarizing wave. In I there is spike inactivation, while the wave maintains long duration and high amplitude although the membrane potential is more depolarized than the control.
383 is more powerful, but more complex. The slow changes of the membrane potential may be due to excitatory or inhibitory inputs coming from neurons activated by bicuculline, while the IPSP flattening, independent of the membrane potential changes, suggests a blockade of the mechanisms mediated by GABA. The long-lasting depolarizing wave could be either a reversal of the IPSP or the prolonged EPSP. However, considering that the IPSP equilibrium potential (Fig. 2B) and the depolarizing wave (Fig. 2D and E) are at the same resting potential (--44 mV), different ionic mechanisms are probably involved in these two responses. Analogous results can be observed in the neuron shown in Fig. 3E and H. Besides, the depolarizing wave persists when the membrane potential in the same neuron shifts from hyperpolarizing (Fig. 3G; - - 6 4 mV) to depolarizing (Fig. 3I; --51 mV) levels as regards the IPSP equilibrium potential (Fig. 3E; - - 6 0 mV). Thus, the evoked depolarizing wave seems to be a prolonged EPSP and not a reversed IPSP. The resistance increase, observed during bicuculline injection, could account in part for the increase in EPSP amplitude and duration. Shank et al. have already described in invertebrates the progressive increase of EPSP amplitude and duration and have related this finding to an increase of the membrane resistance due to the non-specific action of bicuculline 12. Finally we emphasize that GABA antagonism at the neuronal level, observed following the systemic injection of these alkaloids, precedes the seizures in the rat caudate nucleus, in contrast to findings by Nicoll in the olfactory bulb 1°. In conclusion, our results show that picrotoxin blocks GABA inhibition in the caudate. It depolarizes the neuron and decreases the IPSP amplitude whi)e spikes appear during the 1PSP. However, these data do not show whether the picrotoxin antagonism is specific or non-specific. The action of bicuculline is different and its effects seem to rule out the possibility that this alkaloid is a specific antagonist of GABA. Supported by C.N.R. Grant No. 74.00147.04.
l BERNARD1,G., MARCIANI,M. G., MOROCUTTI,C., AND GIACOMINI,P., The action of GABA on rat caudate neurons recorded intracellularly, Brain Research, 92 0975) 511 515. 2 CURTIS,D. R., DUGGAN,A. W., FELIX, D., AND JOHNSTON,G. A. R., GABA, bicuculline and central inhibition, Nature (Lond.), 226 (1970) 1222-1224. 3 CURTIS,D. R., DUGGAN,A. W., FELIX,D., ANDJOHNSTON,G. A. R., Bicuculline, an antagonist of GABA and synaptic inhibition in the spinal cord of the cat, Brain Research, 32 (1971) 69 96.
4 CURTIS,D. R., DUGGAN,A. W., FELIX,D., JOHNSTON,G. A. R., ANDMCLENNAN,H., Antagonism between bicuculline and GABA in the cat brain, Brain Research, 33 (1971) 57-73. 5 FIFKOVA,E., AND MARSALA, J., Stereotaxic atlas for the rat. In J. BURE~, M. PETRAN AND J. ZAClJAR (Eds.), Electrophysiological Methods in Biological Research, Academic Press, New York, 1967, p. 653. 6 GALINDO,A., GABA-picrotoxin interaction in the mammalian CNS, Brain Research, 14 (1969) 763-767. 7 GODFRAIND,J. M., KRNJEVI(~,K., AND PUMAINR., Doubtful value of bicuculline as a specific antagonist of GABA, Nature (Land.), 228 (1970) 675 676. 8 HILL,R. G., SIMMONDS,M. A., ANDSTRAUGHAN,D. W., A comparative study of some convulsant substances as GABA antagonists in the feline cerebral cortex, Brit. J. Pharmacol. 49 (1973) 37-51.
384 9 MORI, S., Some observations on the fine structure of the corpus striatum of the rat brain, Z, Zellfi~rseh., 70 (1966) 461 488. 10 NIcOLL, R. A., Pharmacological evidence for GABA as the transmitter in the granule celt inhibition in the olfactory bulb, Brain Research, 35 (1971) 137-149. 11 OSATA, K., AND HIGHSX~I~, S. M., Blocking by picrotoxin of both vestibulary inhibition and G A B A action on rabbit oculomotor neurons, Brain Research, 18 (1970) 538-541. 12 SHANK, R. P., PONG, S. F., FREEMAN, A. R., AND GRAHAM, L. T., Bicuculline and picrotoxin as antagonists of 3,-aminobutyrate and neuromuscular inhibition in the lobster, Brain Research, 72 (1974) 71-78. 13 STRAUGHAN, D. W., Convulsant drugs: amino acid antagonism and central inhibition, Neuropharmacology, 13 (1974) 495-508. 14 STRAUGHAN, D. W., NEA~,, M. J., SIMMONDS, M. A., COLLINS, G. C. S., ANO HILL, R. G., Evaluation of bicuculline as a GABA antagonist, Nature (Lond.), 223 (1971) 352 354.