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Anticonvulsant effects of GABA elevation in the deep prepiriform cortex
102
Epilepsy Res.. 1 (1987) 102-106
Elsevier ERS 00120
Anticonvulsant effects of GABA elevation in the deep prepiriform cortex
Salvatore Piredda, Mark Pavlick and Karen Gale Department of Pharmacology, Georgetown University, Schools of Medicine and Dentistry, Washington, DC 20007 (U. S. A. )
(Received 6 August 1986; accepted 8 September 1986) Key words: Clonic seizures; Tonic seizures; Bicuculline; GABA deficit; Gamma-vinyl GABA
Microinjection of gamma-vinyl GABA (GVG), a GABA elevating agent, into a discrete region of the deep prepiriform cortex elevated local GABA levels nearly 4-fold and exerted an anticonvulsant action against seizures induced by intravenous injection of the GABA antagonist, bicuculline, but was ineffective against seizures induced by maximal electroshock. This, together with a previous finding that blockade of GABA transmission in the deep prepiriform cortex induces convulsions, suggests that this area may be crucial, if not primarily responsible, for the genesis of clonic seizures resulting from a deficit in GABA function. INTRODUCTION The inhibitory neurotransmitter, g a m m a - a m i n o butyric acid ( G A B A ) , plays an important role in convulsant 3'92°'21 and anticonvulsant 5'H'12 activity. The substantia nigra has been shown to be a site at which G A B A agonists exert anticonvulsant effects, probably by interfering with seizure propagation s. H o w e v e r , the fact that the substantia nigra is not a site from which seizures can be initiated in response to G A B A antagonists suggests that there must be at least one other site in the brain where changes in G A B A transmission are critical for triggering seizures 4. Recently, we identified a discrete site located within the deep prepiriform cortex of the rat from which bilateral m o t o r seizures are elicited by the Correspondence to: Dr. K. Gale, Department of Pharmacology, Georgetown University, Schools of Medicine and Dentistry, Washington, DC 20007, U.S.A.
direct application of very small amounts of the GABA receptor antagonist, bicucullinelS'lT; we have referred to this site as ' a r e a tempestas' (AT) 6. In addition, G A B A transmission at this site may function to inhibit the generation of seizures16; as such, it may be crucial for the development of seizures in response to a deficiency in G A B A transmission. Accordingly, e n h a n c e m e n t of G A B A transmission in A T should attenuate G A B A antagonist-induced seizures. To test this hypothesis, we examined whether elevation of G A B A in A T could prevent seizures induced by the systemic administration of bicuculline. Seizures that are independent of initiating mechanisms in A T might, on the other hand, not be influenced by G A B A elevation at AT. To explore this, we also examined the effect of G A B A elevation in A T on seizures induced by maximal electroshock (MES). MES seizures do not depend upon forebrain mechanisms for either their initiation or progression t'19 but, instead, a p p e a r dependent
0920-1211/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
103 upon brain-stem circuitry 2"19. To elevate G A B A we microinjected g a m m a - v i n y l - G A B A ( G V G ) into A T bilaterally. G V G , an irreversible inhibitor of G A B A transaminase, causes a long-lasting elevation of G A B A in the vicinity of injection when directly applied to brain tissue 8.
rats were then assigned to control and treatment ( G V G ) groups so that the groups had c o m p a r a b l e distributions of T H E durations. In these rats, the following stereotaxic coordinates were used for A T (with the incisor bar 2.4 m m below the interaural line): 2.6 m m anterior to bregma, 3 m m lateral to the midline, and 6 m m below dura.
METHODS RESULTS AND DISCUSSION Male S p r a g u e - D a w l e y rats weighing 350-370 g were used for the first series of studies with bicuculline-induced seizures. The rats were anesthetized with ether and placed in a David K o p f stereotaxic apparatus. G V G was dissolved in saline (10 /~g//~l) and 0.5/~1 was infused bilaterally into A T via a 26-gauge stainless steel cannula at a rate of 0.125 ~l/min. Controls received saline microinjections into AT. The stereotaxic coordinates for A T , when the incisor bar was 5 m m above the interaural line, were the following: 4 m m anterior to bregma, 3 m m lateral to the midline, and 6.5 m m below dura. Injection sites were verified histologically; data presented are from animals in which injections were located within a 0.5 m m radius of the site as previously defined on the basis of response to chemoconvulsants 15. Chemoconvulsant seizure response was evaluated by measuring the severity of seizures produced by the rapid injection of bicuculline into the lateral tail vein of unrestrained rats. A numerical scoring system was used to rate the seizures (see legend to Table I). A separate group of rats, treated with G V G infusions in A T as described above, was not tested with bicuculline but was, instead, killed at 6 h for m e a s u r e m e n t of G A B A levels in selected brain regions. G A B A was assayed according to the method of O k a d a et al.14 with the omission of the 60 °C heating step. MES seizures were induced by means of a Wahlquist apparatus as designed by W o o d b u r y and Davenport22; the 60 Hz, 150 m A current was delivered for 0.2 sec via corneal electrodes. Tonic hind limb extension ( T H E ) , the endpoint in this test, was timed with a stopwatch and its duration was recorded. Rats weighing 120-150 g were screened 24 h prior to each experiment and animals with T H E durations of 4.0 sec or less were eliminated (this represented less than 20% of the rats). The
The effect of microinjections of G V G placed bilaterally into the A T against generalized seizures induced by bicuculline is shown in Table I. Anticonvulsant activity, reflected in a significant decrease in mean seizure score and a decreased incidence of m a j o r clonic and/or tonic seizures, was
TABLE I Anticonvulsant effect of GVG in A T against seizures induced by intravenous bicuculline a Score b
No. of rats showing major seizures (score >12)
Mean
Median Mode
13 13
2.2 1.3"
2 1
3 1
9 2
Cont GVG
12 11
2.0 1.2"
2 1
3 1
7 3
96 hc Cont GVG
7 7
2.7 2.5
3 3
3 3
6 6
6 hc
Cont GVG 24 h c
a
Bicuculline was dissolved in a small volume of 0.1 N HCI, then diluted with saline to a concentration of 0.37 mg/ml. The pH was adjusted to 5.7 with NaOH and the solution was kept
on ice during the experiments. The dose of bicuculline (0.37 mg/kg, i.v.) was chosen in order to produce clonic seizures in all control rats (cont). b Seizure severity was rated using the following scale: 0 = no seizure activity; 0.5 = facial myoclonus and forepaw myoclonus; 1.0 = clonic seizure, lasting at least 15 sec and accompanied by body twist; 2.0 = explosive cionic seizure with wild running; 3.0 = tonic forelimb extension; 4.0 = tonic hind limb extension (THE). c Testing was done at 6, 24, and 96 h after bilateral microinjection of GVG or an equal volume of saline into AT. * Significantly different from saline-treated controls; MannWhitney U test for non-parametric data (P < 0.05).
104 obtained at 6 h; this effect was still present at 24 h. By 96 h after G V G infusion, complete recovery of normal seizure responses was obtained (Table I). This is in agreement with the time course of functional recovery observed after the same dose of G V G placed in substantia nigra s. G V G microinjections placed more than 1 mm away from the A T did not attenuate bicuculline-induced convulsions; it appeared that the G V G injections had to be correctly placed bilaterally in A T in order to be effective. In comparison to saline-injected controls, rats receiving 5 a g of G V G into A T showed nearly a 4fold elevation of G A B A in the vicinity of the injection site ( G A B A nmol/mg prot + S.E.M.: controls, 38.5 + 4.4; GVG-treated, 143.5 + 16.9). On the other hand, no significant elevation of G A B A was observed in amygdala (controls, 29.2 + 2.2; GVG-treated, 32.2 + 2.6), hippocampus (controls, 21.0 + 2.3; GVG-treated, 25.8 + 3.5) or substantia nigra (controls, 88.0 + 8.3; G V G treated, 98.8 + 6.6), indicating that the anticonvulsant effect that we have obtained does not involve a direct action of G V G on any of these latter regions. As shown in Fig. 1, at 6 h after G V G (10/~g bilaterally into AT), there was no effect on duration or
Control C] GVG [ ]
~'
!j
6 hr~
MES
BIC
Fig. 1. Comparison of the effect of G V G in A T on seizures induced by bicuculline (BIC) and MES. The values for MES (means + S.E. for 7-11 rats) represent the duration of the T H E in seconds. T h e values for bicuculline ( m e a n s + S.E.) are derived from the scale used to rate the severity of seizure (see Table I legend). A balanced cross-over design was employed so that rats in group A (left side of graph) were tested with M E S at 6 h (after microinjection), and with BIC at 24 h; rats in group B (right side of graph) were tested in the reverse order. * Significantly different from the control value: Student's t test for MES; M a n n - W h i t n e y U test for bicuculline values, P < 0.05.
incidence of T H E induced by MES; a second group of rats tested in parallel showed significant protection against bicuculline seizures. The dissociation of the effect of G V G against the two kinds of seizures was verified using a cross-over design. Rats which had been tested with MES at 6 h after G V G , were tested with bicuculline at 24 h; conversely, the rats which had been tested with bicuculline at 6 h were tested with MES at 24 h. As can be seen in Fig. 1, the results obtained at 24 h show the same dissociation between bicuculline and MES seizures as was evident at 6 h after G V G microinjection. In this case, the same rats that had been protected from bicuculline at 6 h showed no significant seizure protection in the MES test at 24 h, as compared to the saline-infused animals. Conversely, the same rats that showed no protection against MES at 6 h showed significant protection from bicuculline at 24 h after infusion of G V G into
AT. Thus, in contrast to the results obtained with bicuculline seizures, no protection against MES seizures was obtained following G V G treatment in AT. A change in G A B A transmission in A T therefore does not appear to be crucial for the generation of MES seizures. These results are consistent with the conclusion of Iadarola and Gale s that MES seizures can be attenuated by G A B A elevation only in the substantia nigra. The latter authors found no effect against MES after forebrain and hindbrain microinjections of high doses of G V G which elevated G A B A several-fold throughout forebrain, pontine and tectal tissue s. Another seizure model that is of interest for evaluating the effect of G A B A elevation in A T is amygdala kindling 7"Is. Because of the marked similarity between the convulsions produced by a blockade in GABAergic transmission in A T and those resulting from kindling in the limbic system, it has been hypothesized that the development of kindled seizures might depend on the facilitation of activity in a neural circuit involving A T ~5. If this is true, an elevation of G A B A activity in A T should antagonize seizures produced by electrical stimulation of the amygdala in kindled rats. Recently, it has been observed that amygdala-kindied generalized seizures and cortical afterdischarge in the rat could be suppressed from 6 h to 5
105 days following microinfusion of GVG (2.5 pg) bilaterally in AT (J.R. Stevens, personal communication). In addition, bilateral application of the G A B A agonist, muscimol into AT, raised the threshold for eliciting seizures in amygdala kindled rats 13. Protection against both amygdala-kindled and bicuculline-induced seizures can also be obtained by application of GVG into the substantia nigra 8'1°. However, as a site of GABA-mediated anticonvulsant activity, the AT differs from the substantia nigra in two important respects. First, anticonvulsant activity in the MES model is obtained with G A B A agonists in nigra 8 and not in AT. Second, although bicuculline seizures can be attenuated by G A B A elevation in either nigra 8 or AT, bicuculline seizures can only be elicited from AT and not from substantia nigra. These differences reflect the distinct roles of these structures in the process of seizure control. On the one hand, the substantia nigra represents a site at which G A B A synapses control the propagation of seizures that must be initiated elsewhere in the brain. In this context, nigral G A B A appears to function as a common constraint on seizures initiated from a variety of loci 4. In contrast, the site that we have localized in the deep prepiriform cortex contains G A B A synapses that can control seizures whose origin probably depends (either directly or indirectly) upon activity within this same locus. The AT represents a site from which not only bicuculline, but convulsants such as excitatory amino acid analogues and muscarinic cholinergic agonists can initiate seizures 15. As the effects of all convulsant agents tested to date in AT are subject to blockade by G A B A agonist application at this site 15, it is
REFERENCES 1 Browning, R.A. and Nelson, D.K., Modification of electroshock and pentylenetetrazol seizure patterns in rats after precollicular transections, Exp. Neurol., 93 (1986) 546-556. 2 Browning, R.A., Turner, F.J., Simonton, R.L. and Bundman, M.C., Effect of midbrain and pontine tegmental lesions on the maximal electroshock pattern in rats, Epilepsia, 22 (1981) 583-594. 3 Frye, G.D., McCown, T. and Breese, G.R., Characterization of susceptibility to audiogenic seizures in ethanol-de-
probable that AT is a crucial site for the development of seizures induced by several kinds of chemoconvulsants. Further studies with additional chemoconvulsants will help define those types of seizures for which antagonism of G A B A activity in the AT may be a crucial epileptogenic mechanism, and for which G A B A elevation in AT may be anticonvulsant. In conclusion, it appears that in the presence of elevated G A B A in AT, the blockade of G A B A receptors throughout the brain has diminished effectiveness for producing clonic seizures. This together with our previous findings 15 suggests that for seizures triggered by interference with GABAmediated inhibition, the AT represents a key site for both the initiation and control of these seizures. Moreover, the control exerted by the AT may not be limited to seizures induced by chemoconvulsants but may include seizures generated in response to kindling in the limbic system. It is possible that the propagation of discharge from limbic regions to AT may be a critical relay in the generation of limbic seizures. In this context, the AT may function as a broadcasting mechanism, triggering generalized seizures in response to stimulation of limbic circuits. ACKNOWLEDGEMENTS Supported by HHS Grant NS20576 and a NIMH Research Scientist Development Award (MH00497) to Karen Gale. Gamma-vinyl G A B A was a gift from Dow-Merrel Centre de Recherche (Strasbourg, France). We thank Dr. J.R. Stevens for her helpful comments and advice.
pendent rats after microinjection of GABA agonists into the inferior colliculus, substantia nigra or medial septum, J. Pharmacol. exp. Ther., 227 (1983) 663-670. 4 Gale, K., Mechanisms of seizure control mediated by gamma-aminobutyric acid: role of substantia nigra, Fed. Proc., 44 (1985) 2214-2424. 5 Gale, K. and Iadarola, M., Seizure protection and increased nerve terminal GABA, Science, 208 (1980) 288-291. 6 Gale, K. and Piredda, S., Identification of a discrete forebrain site responsible for the genesis of chemically-induced seizures. In: G. Nistico et al. (Eds.), Neurotransmitters, Sei-
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7
8
9
10
11 12
13
14
zures and Epilepsy, 111, Raven Press, New York, 1986, pp. 205-219. Goddard, G.V., Mclntyre, D.C. and Leech, C.K., A permanent change in brain function resulting from daily electrical stimulation, Exp. NeuroL, 25 (1969) 295-330. Iadarola, M.J. and Gale, K., Substantia nigra: site of anticonvulsant activity mediated by gamma-aminobutyric acid, Science, 218 (1982) 1237-1240. Krnjevi6, K., GABA-mediated inhibitory mechanisms in relation to epileptic discharge. In: H.H. Jaspers and N.M. Van Gelder (Eds.), Basic Mechanisms of Neuronal Hyperexcitability, Alan R. Liss, New York, 1983, pp. 249-280. Le Gal la Salle, G., Kijima, M. and Feldblum, S., Abortive amygdaloid kindled seizures following microinjection of gamma-vinyl-GABA in the vicinity of substantia nigra in rats, Neurosci. Lett., 36 (1983) 69-74. Meldrum, B.S., Pharmacology of GABA, Neuropharmacology, 5 (1982) 293-316. Meldrum, B.S., GABA and other amino acids. In: H.H. Frey and D. Janz (Eds.), Antiepileptic Drugs, Springer, Berlin, 1985, pp. 153-188. Morimoto, K., Dragunow, M. and Goddard, G.V., Deep prepyriform cortex kindling and its relation to amygdala kindling in the rat, Exp. Neurol., 94 (1986) in press. Okada, Y., Nitsch-Hassler, C., Kim, J.S., Bak, l.J. and Hassler, R., Role of gamma-aminobutyric acid (GABA) in extrapyramidal motor system. 1. Regional distribution of G A B A in rabbits, rat, guinea pig and baboon CNS, Exp. Brain Res., 13 (1971) 514-518.
15 Piredda, S. and Gale, K., A crucial epileptogenic site in the deep prepiriform cortex, Nature (Lond.), 317 (1985) 623-625. 16 Piredda, S. and Gale, K., Anticonvulsant action of 2-amino-7-phosphonoheptanoic acid and muscimol in the deep prepiriform cortex, Europ. J. Pharmacol., 120 (1986) 115-118. 17 Piredda, S., Lim, C.R. and Gale, K., Intracerebral site of convulsant action of bicuculline. Life Sci., 36 (1985) 1295-1298. 18 Racine, R.J., Modification of seizure activity by electrical stimulation. II. Motor seizure, Electroenceph. clin. Neurophysiol., 32 (1972) 281-294. 19 Tanaka, K. and Mishima, O., The localization of the center dealing with the tonic extensor seizure of electroshock, Jap. J. Pharmacol., 3 (1953) 6-9. 20 Tower, D.B., GABA and seizures: clinical correlates in man. In: E. Roberts, T.N. Chase and D.B. Tower (Eds.), GABA in Nervous Function, Raven Press, New York. 1976, p. 461. 21 Woodbury, D.M., Convulsant drugs: mechanisms of action. In: G.H. Glaser, J.K. Penry and D.M. Woodbury (Eds.), Antiepileptic Drugs. Mechanisms of Action. Adv. Neurol., Vol. 217, Raven Press, New York, 1980, pp. 249-303. 22 Woodbury, L.A. and Davenport, V.D., Design and use of a new electroshock seizure apparatus and analysis of factors altering seizure threshold and pattern, Arch. int. Pharmacodyn. Ther., 92 (1952) 97-107.
Epilepsy Res.. 1 (1987) 102-106
Elsevier ERS 00120
Anticonvulsant effects of GABA elevation in the deep prepiriform cortex
Salvatore Piredda, Mark Pavlick and Karen Gale Department of Pharmacology, Georgetown University, Schools of Medicine and Dentistry, Washington, DC 20007 (U. S. A. )
(Received 6 August 1986; accepted 8 September 1986) Key words: Clonic seizures; Tonic seizures; Bicuculline; GABA deficit; Gamma-vinyl GABA
Microinjection of gamma-vinyl GABA (GVG), a GABA elevating agent, into a discrete region of the deep prepiriform cortex elevated local GABA levels nearly 4-fold and exerted an anticonvulsant action against seizures induced by intravenous injection of the GABA antagonist, bicuculline, but was ineffective against seizures induced by maximal electroshock. This, together with a previous finding that blockade of GABA transmission in the deep prepiriform cortex induces convulsions, suggests that this area may be crucial, if not primarily responsible, for the genesis of clonic seizures resulting from a deficit in GABA function. INTRODUCTION The inhibitory neurotransmitter, g a m m a - a m i n o butyric acid ( G A B A ) , plays an important role in convulsant 3'92°'21 and anticonvulsant 5'H'12 activity. The substantia nigra has been shown to be a site at which G A B A agonists exert anticonvulsant effects, probably by interfering with seizure propagation s. H o w e v e r , the fact that the substantia nigra is not a site from which seizures can be initiated in response to G A B A antagonists suggests that there must be at least one other site in the brain where changes in G A B A transmission are critical for triggering seizures 4. Recently, we identified a discrete site located within the deep prepiriform cortex of the rat from which bilateral m o t o r seizures are elicited by the Correspondence to: Dr. K. Gale, Department of Pharmacology, Georgetown University, Schools of Medicine and Dentistry, Washington, DC 20007, U.S.A.
direct application of very small amounts of the GABA receptor antagonist, bicucullinelS'lT; we have referred to this site as ' a r e a tempestas' (AT) 6. In addition, G A B A transmission at this site may function to inhibit the generation of seizures16; as such, it may be crucial for the development of seizures in response to a deficiency in G A B A transmission. Accordingly, e n h a n c e m e n t of G A B A transmission in A T should attenuate G A B A antagonist-induced seizures. To test this hypothesis, we examined whether elevation of G A B A in A T could prevent seizures induced by the systemic administration of bicuculline. Seizures that are independent of initiating mechanisms in A T might, on the other hand, not be influenced by G A B A elevation at AT. To explore this, we also examined the effect of G A B A elevation in A T on seizures induced by maximal electroshock (MES). MES seizures do not depend upon forebrain mechanisms for either their initiation or progression t'19 but, instead, a p p e a r dependent
0920-1211/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
103 upon brain-stem circuitry 2"19. To elevate G A B A we microinjected g a m m a - v i n y l - G A B A ( G V G ) into A T bilaterally. G V G , an irreversible inhibitor of G A B A transaminase, causes a long-lasting elevation of G A B A in the vicinity of injection when directly applied to brain tissue 8.
rats were then assigned to control and treatment ( G V G ) groups so that the groups had c o m p a r a b l e distributions of T H E durations. In these rats, the following stereotaxic coordinates were used for A T (with the incisor bar 2.4 m m below the interaural line): 2.6 m m anterior to bregma, 3 m m lateral to the midline, and 6 m m below dura.
METHODS RESULTS AND DISCUSSION Male S p r a g u e - D a w l e y rats weighing 350-370 g were used for the first series of studies with bicuculline-induced seizures. The rats were anesthetized with ether and placed in a David K o p f stereotaxic apparatus. G V G was dissolved in saline (10 /~g//~l) and 0.5/~1 was infused bilaterally into A T via a 26-gauge stainless steel cannula at a rate of 0.125 ~l/min. Controls received saline microinjections into AT. The stereotaxic coordinates for A T , when the incisor bar was 5 m m above the interaural line, were the following: 4 m m anterior to bregma, 3 m m lateral to the midline, and 6.5 m m below dura. Injection sites were verified histologically; data presented are from animals in which injections were located within a 0.5 m m radius of the site as previously defined on the basis of response to chemoconvulsants 15. Chemoconvulsant seizure response was evaluated by measuring the severity of seizures produced by the rapid injection of bicuculline into the lateral tail vein of unrestrained rats. A numerical scoring system was used to rate the seizures (see legend to Table I). A separate group of rats, treated with G V G infusions in A T as described above, was not tested with bicuculline but was, instead, killed at 6 h for m e a s u r e m e n t of G A B A levels in selected brain regions. G A B A was assayed according to the method of O k a d a et al.14 with the omission of the 60 °C heating step. MES seizures were induced by means of a Wahlquist apparatus as designed by W o o d b u r y and Davenport22; the 60 Hz, 150 m A current was delivered for 0.2 sec via corneal electrodes. Tonic hind limb extension ( T H E ) , the endpoint in this test, was timed with a stopwatch and its duration was recorded. Rats weighing 120-150 g were screened 24 h prior to each experiment and animals with T H E durations of 4.0 sec or less were eliminated (this represented less than 20% of the rats). The
The effect of microinjections of G V G placed bilaterally into the A T against generalized seizures induced by bicuculline is shown in Table I. Anticonvulsant activity, reflected in a significant decrease in mean seizure score and a decreased incidence of m a j o r clonic and/or tonic seizures, was
TABLE I Anticonvulsant effect of GVG in A T against seizures induced by intravenous bicuculline a Score b
No. of rats showing major seizures (score >12)
Mean
Median Mode
13 13
2.2 1.3"
2 1
3 1
9 2
Cont GVG
12 11
2.0 1.2"
2 1
3 1
7 3
96 hc Cont GVG
7 7
2.7 2.5
3 3
3 3
6 6
6 hc
Cont GVG 24 h c
a
Bicuculline was dissolved in a small volume of 0.1 N HCI, then diluted with saline to a concentration of 0.37 mg/ml. The pH was adjusted to 5.7 with NaOH and the solution was kept
on ice during the experiments. The dose of bicuculline (0.37 mg/kg, i.v.) was chosen in order to produce clonic seizures in all control rats (cont). b Seizure severity was rated using the following scale: 0 = no seizure activity; 0.5 = facial myoclonus and forepaw myoclonus; 1.0 = clonic seizure, lasting at least 15 sec and accompanied by body twist; 2.0 = explosive cionic seizure with wild running; 3.0 = tonic forelimb extension; 4.0 = tonic hind limb extension (THE). c Testing was done at 6, 24, and 96 h after bilateral microinjection of GVG or an equal volume of saline into AT. * Significantly different from saline-treated controls; MannWhitney U test for non-parametric data (P < 0.05).
104 obtained at 6 h; this effect was still present at 24 h. By 96 h after G V G infusion, complete recovery of normal seizure responses was obtained (Table I). This is in agreement with the time course of functional recovery observed after the same dose of G V G placed in substantia nigra s. G V G microinjections placed more than 1 mm away from the A T did not attenuate bicuculline-induced convulsions; it appeared that the G V G injections had to be correctly placed bilaterally in A T in order to be effective. In comparison to saline-injected controls, rats receiving 5 a g of G V G into A T showed nearly a 4fold elevation of G A B A in the vicinity of the injection site ( G A B A nmol/mg prot + S.E.M.: controls, 38.5 + 4.4; GVG-treated, 143.5 + 16.9). On the other hand, no significant elevation of G A B A was observed in amygdala (controls, 29.2 + 2.2; GVG-treated, 32.2 + 2.6), hippocampus (controls, 21.0 + 2.3; GVG-treated, 25.8 + 3.5) or substantia nigra (controls, 88.0 + 8.3; G V G treated, 98.8 + 6.6), indicating that the anticonvulsant effect that we have obtained does not involve a direct action of G V G on any of these latter regions. As shown in Fig. 1, at 6 h after G V G (10/~g bilaterally into AT), there was no effect on duration or
Control C] GVG [ ]
~'
!j
6 hr~
MES
BIC
Fig. 1. Comparison of the effect of G V G in A T on seizures induced by bicuculline (BIC) and MES. The values for MES (means + S.E. for 7-11 rats) represent the duration of the T H E in seconds. T h e values for bicuculline ( m e a n s + S.E.) are derived from the scale used to rate the severity of seizure (see Table I legend). A balanced cross-over design was employed so that rats in group A (left side of graph) were tested with M E S at 6 h (after microinjection), and with BIC at 24 h; rats in group B (right side of graph) were tested in the reverse order. * Significantly different from the control value: Student's t test for MES; M a n n - W h i t n e y U test for bicuculline values, P < 0.05.
incidence of T H E induced by MES; a second group of rats tested in parallel showed significant protection against bicuculline seizures. The dissociation of the effect of G V G against the two kinds of seizures was verified using a cross-over design. Rats which had been tested with MES at 6 h after G V G , were tested with bicuculline at 24 h; conversely, the rats which had been tested with bicuculline at 6 h were tested with MES at 24 h. As can be seen in Fig. 1, the results obtained at 24 h show the same dissociation between bicuculline and MES seizures as was evident at 6 h after G V G microinjection. In this case, the same rats that had been protected from bicuculline at 6 h showed no significant seizure protection in the MES test at 24 h, as compared to the saline-infused animals. Conversely, the same rats that showed no protection against MES at 6 h showed significant protection from bicuculline at 24 h after infusion of G V G into
AT. Thus, in contrast to the results obtained with bicuculline seizures, no protection against MES seizures was obtained following G V G treatment in AT. A change in G A B A transmission in A T therefore does not appear to be crucial for the generation of MES seizures. These results are consistent with the conclusion of Iadarola and Gale s that MES seizures can be attenuated by G A B A elevation only in the substantia nigra. The latter authors found no effect against MES after forebrain and hindbrain microinjections of high doses of G V G which elevated G A B A several-fold throughout forebrain, pontine and tectal tissue s. Another seizure model that is of interest for evaluating the effect of G A B A elevation in A T is amygdala kindling 7"Is. Because of the marked similarity between the convulsions produced by a blockade in GABAergic transmission in A T and those resulting from kindling in the limbic system, it has been hypothesized that the development of kindled seizures might depend on the facilitation of activity in a neural circuit involving A T ~5. If this is true, an elevation of G A B A activity in A T should antagonize seizures produced by electrical stimulation of the amygdala in kindled rats. Recently, it has been observed that amygdala-kindied generalized seizures and cortical afterdischarge in the rat could be suppressed from 6 h to 5
105 days following microinfusion of GVG (2.5 pg) bilaterally in AT (J.R. Stevens, personal communication). In addition, bilateral application of the G A B A agonist, muscimol into AT, raised the threshold for eliciting seizures in amygdala kindled rats 13. Protection against both amygdala-kindled and bicuculline-induced seizures can also be obtained by application of GVG into the substantia nigra 8'1°. However, as a site of GABA-mediated anticonvulsant activity, the AT differs from the substantia nigra in two important respects. First, anticonvulsant activity in the MES model is obtained with G A B A agonists in nigra 8 and not in AT. Second, although bicuculline seizures can be attenuated by G A B A elevation in either nigra 8 or AT, bicuculline seizures can only be elicited from AT and not from substantia nigra. These differences reflect the distinct roles of these structures in the process of seizure control. On the one hand, the substantia nigra represents a site at which G A B A synapses control the propagation of seizures that must be initiated elsewhere in the brain. In this context, nigral G A B A appears to function as a common constraint on seizures initiated from a variety of loci 4. In contrast, the site that we have localized in the deep prepiriform cortex contains G A B A synapses that can control seizures whose origin probably depends (either directly or indirectly) upon activity within this same locus. The AT represents a site from which not only bicuculline, but convulsants such as excitatory amino acid analogues and muscarinic cholinergic agonists can initiate seizures 15. As the effects of all convulsant agents tested to date in AT are subject to blockade by G A B A agonist application at this site 15, it is
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probable that AT is a crucial site for the development of seizures induced by several kinds of chemoconvulsants. Further studies with additional chemoconvulsants will help define those types of seizures for which antagonism of G A B A activity in the AT may be a crucial epileptogenic mechanism, and for which G A B A elevation in AT may be anticonvulsant. In conclusion, it appears that in the presence of elevated G A B A in AT, the blockade of G A B A receptors throughout the brain has diminished effectiveness for producing clonic seizures. This together with our previous findings 15 suggests that for seizures triggered by interference with GABAmediated inhibition, the AT represents a key site for both the initiation and control of these seizures. Moreover, the control exerted by the AT may not be limited to seizures induced by chemoconvulsants but may include seizures generated in response to kindling in the limbic system. It is possible that the propagation of discharge from limbic regions to AT may be a critical relay in the generation of limbic seizures. In this context, the AT may function as a broadcasting mechanism, triggering generalized seizures in response to stimulation of limbic circuits. ACKNOWLEDGEMENTS Supported by HHS Grant NS20576 and a NIMH Research Scientist Development Award (MH00497) to Karen Gale. Gamma-vinyl G A B A was a gift from Dow-Merrel Centre de Recherche (Strasbourg, France). We thank Dr. J.R. Stevens for her helpful comments and advice.
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