- Email: [email protected]
Anticonvulsant activity of the glial-selective GABA uptake inhibitor, THPO
NeuropharmacologyVol.
22, pp. 139-142, 1983
0028-3908/83/010139-0450t00/0 Pergamon Press Ltd
Printed in Great Britain
ANTI--ANT
ACTIVITY OF TI~ ( ~ I A L - E E ~
CdkBA UFrAI(E INHIBITOR, THPO.
J.D. Wood, 1 D.D. Johnson 2, P. Krogsgaard-Larsen 3 and A. Schousboe 4 3_~partment of Biochemistry, University of Saskatchewan, Saskatoon, Canada; partment of Pharmacology, University of Saskatchewan, Saskatoon, Canada; Dep@rh~ent of Chemistry BC, Royal Danish School of Pharmacy, Copenhagen, Dermark; and=Department of Biochemistry A, Panum Institute, University of Copenhagen, Copenhagen, Dermark.
(Ae~pted 26 O~tobg~t 79~2) Stmmary: The intramuscular administration of 4,5,6,7-tetrahydroisoxazolo[4,5-_c] pyridin-3-ol (THPO) delayed the onset of isonicotinic acid hydrazide-induced seizures in very young chicks but not ~n adult mice, the difference being due to the state of development of the blood-brain-barrier which controls access of the drug to the brain tissue. THPO was also effective in preventing seizures induced in epileptic chicks by intermittent photic stimulation. The anticonvulsant action after combined administration of THPO and gabaculine, an inhibitor of GABA-e-oxoglutarate aminotransferase activity, was no greater than the anticonvulsant action of gabaculine alone. The termination of GABA function in the synaptic cleft by transport of the amino acid into adjacent cellular structures (Iversen and Kelly, 1975) raises the possibility that the administration of drugs which "block this uptake may enhance the GABA-induced synaptic inhibition with ooncomitant anticonvulsant effects. Although the potent GABA uptake inhibitor, nipecotic acid, does not penetrate the blood-brain-barrier, its ethyl ester does penetrate into the brain tissue where it is hydrolyzed to the free amino acid with a resultant anticonvulsant action (Frey, Popp and Loscher, 1979). However, nipecotic acid inhibits the uptake of GABA into both neurones and glial cells (Schousboe, Larsson, Hertz and Krogsgaard-Larsen, 1981) and the advantage gained by increasing the concentration of Cd%BA in the synaptic cleft may be partially offset by a decreased content of CABA in the nerve terminals leading to a decrease {n the amount of CdH3A released into the cleft. It should theoretically be possible to obviate this drawback by using an uptake inhibitor which is specific for glial cells since this type of drug would elevate the concentration of GABA in the synaptic cleft while still allowing reuptake of GABA into the nerve terminals. 4,5,6,7-tetrahydroisoxazolo[4,5-c_]pyridin-3-ol (THPO) (Fig. I) is such a og,lc~und (Schousboe, Larsson, Hertz and Krogsgaard-Larsen, 1981) and its anticonvulsant action has been tested and is reported here.
HOF~ .O HsC_~v_)__~O HO~O
HO-~,~N,
HN.v~J
HN~v~0
(RS)-Nipecotic Acid Fig. i.
HN~
HN~ '~OH
Ethyl (3RS,/.SR)-4-Mydtoxy[R)-Nipecotate nipecoticAcid THPO
H~O <~0 HNS
H2N
Momo-8-proline Gabocu[ine
Structural relationship of the test drugs. METHODS
Male Swiss mice weighing 25-30g and White Leghorn oockerels aged 3- and 21-days old respectively were injected intramuscularly with the drugs in the dosage levels indicated in Table i. The animals were observed continuously for 90 rain. after the intramuscular injection of the isonicotinic acid hydrazide (INH) and the time to the onset of generalized convulsions was recorded. Three-day-old chicks of either sex from a flock of epileptic chicken maintained at the University of Saskatchewan were also used in the present study. These birds have a high seizure susceptibility due to an autosomal recessive gene, and homozygotes are highly sensitive to intermittent photic stimulation (IPS) (Crichlow and Crawford, 1974). The birds were placed in pairs (i control and 1 treated) in a bell jar with a strobe light placed at a distance of 12" , exposed to IPS (14 flashes/sec.) for two minutes, and the oocurence of seizures recorded. 4,5,6,7-Tetrahydroisoxazolo [4,5-c_]pyridin-3-ol (THPO) (Krogsgaard-Larsen and Hjeds, 1974), (RS)-pyrrolidine-3-acetic acid (homo-@-proline) (Thorbek, Hjeds and Schat~flaurg, 1981), ethyl (R)-nipecotate (Akkerman, De Jongh and Veldstra, 1951), (3RS,dSR)-4hydroxynipecotic acid (cis-4-hydroxynipecotic acid) (Krogsgaard-Larsen, Thyssen and 139
140
Preliminary Notes
Schamburg, 1978) and gabaculine (Wood, Geddes, Tsui and Kurylo, 1982) were synthesized by previously published procedures. Statistical analysis was carried out using the Student t test (Table i) and Fisher's exact test for independence in 2 x 2 tables Cfable 2).
Table I.
Anticonvulsant action of THPO and other drugs against INH-induced seizures.
Species
Drug (mmol/kg)
Time to convulsions (rain)
Mouse
Control
23.1 + 0.6 (36)
THPO (0.5)
24.1 + 1.0 (12)
THPO (I.0)
23.5 + 0.6 (12)
Ethyl (R)-nipecotate (0.5)
45.5 + 5.4* (12)
Gabaculine (0.5)
53.6 + 1.8" (24)
(0.5) + gabaculine (0.5)
58.8 + 3.6* (12)
Ethyl (R)-nipecotate (0.5) + gabaculine (0.5)
45.5 + 1.5" (12)
Chick (21 days)
Control
24.5 + 1.0 (16)
THPO (i.0)
30.9 + i.i* (16)
Chick (3 days)
Control
25.6 + 0.6 (64)
Hcmo-~-proline (i.0)
24.0 + 1.2 (12)
qis-4-hydromynipecotic acid (i.0)
28.9 ~ 1.5 (15)
(RS)-nipecotic acid (2.0)
24.3 + 0.8 (16)
Ethyl (R)-nipecotate (i.0)
32.3 + 1.8" (14)
THPO (i.0)
44.5 + 1.9, (16)
Gabaculine (0.5)
66.8 + 3.5* (8/16)
THPO (i.0) + gabaculine (0.5)
70.2 + 3.5* (8/16)
The drugs were injected intramuscularlyone hour prior to the intramuscu]ar administration of INH (4.0 mmol/kg). Convulsion times are the mean + S E M for the nt~mber of animals given in parenthesis. Only 8/16 animals convulsed in the last two groups and these means are therefore calculated on n = 8. *Significantly different from controls (P < 0.01). Table 2.
Anticonvulsant action of %~4PO against seizures induced in epileptic chicks by intermittent photic stimulation. Incidence of seizures at various times after injection. lh 2h 4h 22h
Control THPO (i.0)
15/24 5/24*
18/24 5/24*
19/24 8/24*
19/24 16/24
*Significantly different from controls at P < 0.003. ~ESULTS The administration of ~FHPO to mice did not delay the onset of INH-induced seizures, in contrast to the effect of ethyl (R)-nipecotate which brought about a doubling of the time to onset of seizures (Table i). The administration of the GABA-e-oxoglutarate aminotransferase (GABA-T) inhibitor, gabaculine, caused a significant delay in the onset of the INH-induced convulsions, but the concurrent administration of the uptake inhibitors did not enhance the gabaculine anticonvulsant action. In contrast to its lack of effect in mice, ~ brought about a significant delay in the onset of INH-induced seizures in both 3-day-old and 21-day-old chicks, but more so in the younge r chicks (Table i). Again, ethyl (R)-nipecotate produced a significant delay in the seizures, but homo-~-proline, cis-4-hydroxynipecotic acid and (RS)-nipecotic acid
Preliminary Notes
14
itself were without effect in the chicks. Although both gabaculine and THPO produced an anticonvulsant effect in chicks, the action of the oombined drug treatment was only slightly better than that of gabaculine itself. THPO also significantly decreased the number of epileptic chicks having convulsions due to intermittent photic stimulation (Table 2). This effect was relatively longlasting, being still observed 4 hours after the administration of THPO. DISCUSSION While the lack of anticonvulsant activity of THPO in mice may simply indicate that the compound is inactive, an alternate possibility is that it does possess anticonvulsant properties per s__eebut does not penetrate the blood-brain-barrier. Since this barrier in very young chicks is less effective in excluding compounds such as GABA (Wood, 1970), the chick model provides an opportunity to test the anticonvulsant efficacy of GABA analogs which do not penetrate a blood-brain-barrier which has reached a more complete stage of development. Use of this mode] in the present study clearly indicates that THPO does possess anticonvulsant properties (Tables 1 and 2) and points to the lack of effect in mice as being due to permeability probl~ns. Supporting evidence for this conclusion is forthcoming from mouse studies in which THPO does not provide protection frQm maximal electroshock and metrazol-induced seizures (unpublished observations), and from the chick studies in which the anticonvulsant action of THPO decreases with the age of the birds as the blood-brain-barrier develops and becomes more efficient (Table i). The lack of antieonvulsant action of homo~-prollne, cis-4-hydro~ynipecotic acid and (RS)-nipecotic acid in 3-day-01d chicks suggests that even in very young birds the blood-brain-barrier is sufficiently developed to exclude these amino carboxylic acids with potent effects on GABA uptake in vitro, particularly since nipecotic acid is known to possess anticonvulsant activity when it is injected directly into the brain (Horton, Collins, Anlezark and Meldr u~, 1979). Since the anticonvulsant actio~ of gabaculine is probably brought about by increased GABA levels, particularly in the nerve endings (Wood, Russell and Kurylo, 1980), and since the antioonvulsant action of THR9 is probably caused by inhibition of the uptake of GABA from the synaptic cleft into glial cells, it seems reasonable to expect an additive effect when the two compounds are administered concurrently. Unexpectedly, the anticonvulsant action of the drug combination was only slightly better than that of gabaculine alone (Table i). The reason for this phenomenon is uncertain but may be due to the THPO interfering with the uptake of gabaculine into the cells, as was observed previously with gabaculine and the GABA uptake inhibitor, ketamine ~kx)d, Geddes, Tsui and Kurylo, 1982). In conclusion, it would seem that glial-selective irthibitors of GABA uptake are promising candidates as antioonvulsant agents, but that the impermeability of the bloodbrain-barrier to these drugs greatly lessens their usefulness. Further research directed towards the development of a glial-se!ective inhibitor which passes readily into the brain would therefore seem a top priority. A~DGEMENT The authors thank Mr. E. Kurylo for his skilled technical assistance. The work was supported by grants MT-3801 and Mr-5893 from the Medical Research Council of Canada and by grants from the Danish Medical Research Concil and the NOVO Foundation. ;~FE~NCES Akkerman, A. M., De Jongh, D. K. and Ve!dstra, H. (1951). Synthetic oxytocics. I. 3-(Piperidyl-(N)-methyl)-indoles and related compounds. Recl. Tray. Chim. Pays-Bas Belg. 70: 899-916. Crichlow, E.C. and Crawford, R.D. (1974). Epileptiform seizures in domestic fowl II. Intermittent ]ight stimulation and electroencephalogram. Canad. J. Physiol. Pharmacol. 52: 424-429. Frey, H.-H., Popp, C. and Loscher, W. (1979). Influence of inhibitors of the high affinity GABA uptake on seizure thresholds in mice. Neuropharmacol. 18: 581-590. Horton, R.W., Collins, J.F., Anlezark, G.M. and Meldrt~, B.S. (1979). Convulsant and anticonvulsant actions in DBA/2 mice of compounds blocking the reuptake of GABA. Eur. J. Pharmacol. 59: 75-83. Iversen, L.L. and Kelly, J.S. (1975). Uptake and metabolism of 7-aminobutyric acid by neurones and glial cells. Biochsm. Pharmacol. 34: 933-938. Krogsgaard-Larsen, P. and Hjeds, H. (1974). Structural analogues of ?-aminoburyric acid (GABA) of the isoxazole enol-betaine type. Synthesis of 5,6,7,8-tetrahydro-4Hisoxazolo [4,5-d] -azepin-3-ol zwitter ion and 4,5,6,7-tetrahydr isoxazol [4,5-c] pyridin-3-ol zwitterion. Acta Chem. Scand. B28: 533-538. Krogsgaard-Larsen, ~., Thyssen, K. and Schau~m/rg, K. (1978). Inhibitors of GABA uptake. Synthesis and --H NMR spectroscopic investigations of guvacine, (3RS, 4SR)-4-hydroxypiperidin~3-carboxylic acid and related compounds. Acta Chem. Scand. B32: 327-334. Schousboe, A., Larsson, O.M., Hertz, L. and Krogsgaard-Larsen, P. (1981) Heterocyclic GABA
42
Preliminary Notes
analogues as selective inhibitors of astroglial C~%BA uptake. In: Amino Acid Tranmmitters (De Feudis, F.V. and Mandel, P., eds.) pp. 135~i145. Thorbek, P., Hjeds, H. and Schaumburg, K. (1981). Syntheses and ~H h~4R spectroscopic investigations of scrne pyrrolidine carboxylic acids designed as potential glial GABA uptake ir[hibitors. Acta Chem. Scand. B35: 473-479. Wood, J.D. (1970). Seizures induced by hyperbaric oxygen and cerebral ¥-eminobutyric acid in chicks during development. J. Neurochem. 17: 573-579. Wood, J.D., Geddes, J.W., Tsui, S-K. and Kurylo, E. (1982). Combined effects of a metabolic inhibitor (gabaculine) and an uptake inhibitor (ketamine) on the T-aminobutyrate system in mouse brain. J. Neurochem. (in press). Wood, J.D., Russell, M.P. and Kurylo, E. (1980). The Y-aminobutyrate content of nerve endings (synaptosomes) in mice after the injection of Y-aminoburyrate-elevating agents: a possible role ~n anticonvulsant activity. J. Neurochem. 35: 125-130.
22, pp. 139-142, 1983
0028-3908/83/010139-0450t00/0 Pergamon Press Ltd
Printed in Great Britain
ANTI--ANT
ACTIVITY OF TI~ ( ~ I A L - E E ~
CdkBA UFrAI(E INHIBITOR, THPO.
J.D. Wood, 1 D.D. Johnson 2, P. Krogsgaard-Larsen 3 and A. Schousboe 4 3_~partment of Biochemistry, University of Saskatchewan, Saskatoon, Canada; partment of Pharmacology, University of Saskatchewan, Saskatoon, Canada; Dep@rh~ent of Chemistry BC, Royal Danish School of Pharmacy, Copenhagen, Dermark; and=Department of Biochemistry A, Panum Institute, University of Copenhagen, Copenhagen, Dermark.
(Ae~pted 26 O~tobg~t 79~2) Stmmary: The intramuscular administration of 4,5,6,7-tetrahydroisoxazolo[4,5-_c] pyridin-3-ol (THPO) delayed the onset of isonicotinic acid hydrazide-induced seizures in very young chicks but not ~n adult mice, the difference being due to the state of development of the blood-brain-barrier which controls access of the drug to the brain tissue. THPO was also effective in preventing seizures induced in epileptic chicks by intermittent photic stimulation. The anticonvulsant action after combined administration of THPO and gabaculine, an inhibitor of GABA-e-oxoglutarate aminotransferase activity, was no greater than the anticonvulsant action of gabaculine alone. The termination of GABA function in the synaptic cleft by transport of the amino acid into adjacent cellular structures (Iversen and Kelly, 1975) raises the possibility that the administration of drugs which "block this uptake may enhance the GABA-induced synaptic inhibition with ooncomitant anticonvulsant effects. Although the potent GABA uptake inhibitor, nipecotic acid, does not penetrate the blood-brain-barrier, its ethyl ester does penetrate into the brain tissue where it is hydrolyzed to the free amino acid with a resultant anticonvulsant action (Frey, Popp and Loscher, 1979). However, nipecotic acid inhibits the uptake of GABA into both neurones and glial cells (Schousboe, Larsson, Hertz and Krogsgaard-Larsen, 1981) and the advantage gained by increasing the concentration of Cd%BA in the synaptic cleft may be partially offset by a decreased content of CABA in the nerve terminals leading to a decrease {n the amount of CdH3A released into the cleft. It should theoretically be possible to obviate this drawback by using an uptake inhibitor which is specific for glial cells since this type of drug would elevate the concentration of GABA in the synaptic cleft while still allowing reuptake of GABA into the nerve terminals. 4,5,6,7-tetrahydroisoxazolo[4,5-c_]pyridin-3-ol (THPO) (Fig. I) is such a og,lc~und (Schousboe, Larsson, Hertz and Krogsgaard-Larsen, 1981) and its anticonvulsant action has been tested and is reported here.
HOF~ .O HsC_~v_)__~O HO~O
HO-~,~N,
HN.v~J
HN~v~0
(RS)-Nipecotic Acid Fig. i.
HN~
HN~ '~OH
Ethyl (3RS,/.SR)-4-Mydtoxy[R)-Nipecotate nipecoticAcid THPO
H~O <~0 HNS
H2N
Momo-8-proline Gabocu[ine
Structural relationship of the test drugs. METHODS
Male Swiss mice weighing 25-30g and White Leghorn oockerels aged 3- and 21-days old respectively were injected intramuscularly with the drugs in the dosage levels indicated in Table i. The animals were observed continuously for 90 rain. after the intramuscular injection of the isonicotinic acid hydrazide (INH) and the time to the onset of generalized convulsions was recorded. Three-day-old chicks of either sex from a flock of epileptic chicken maintained at the University of Saskatchewan were also used in the present study. These birds have a high seizure susceptibility due to an autosomal recessive gene, and homozygotes are highly sensitive to intermittent photic stimulation (IPS) (Crichlow and Crawford, 1974). The birds were placed in pairs (i control and 1 treated) in a bell jar with a strobe light placed at a distance of 12" , exposed to IPS (14 flashes/sec.) for two minutes, and the oocurence of seizures recorded. 4,5,6,7-Tetrahydroisoxazolo [4,5-c_]pyridin-3-ol (THPO) (Krogsgaard-Larsen and Hjeds, 1974), (RS)-pyrrolidine-3-acetic acid (homo-@-proline) (Thorbek, Hjeds and Schat~flaurg, 1981), ethyl (R)-nipecotate (Akkerman, De Jongh and Veldstra, 1951), (3RS,dSR)-4hydroxynipecotic acid (cis-4-hydroxynipecotic acid) (Krogsgaard-Larsen, Thyssen and 139
140
Preliminary Notes
Schamburg, 1978) and gabaculine (Wood, Geddes, Tsui and Kurylo, 1982) were synthesized by previously published procedures. Statistical analysis was carried out using the Student t test (Table i) and Fisher's exact test for independence in 2 x 2 tables Cfable 2).
Table I.
Anticonvulsant action of THPO and other drugs against INH-induced seizures.
Species
Drug (mmol/kg)
Time to convulsions (rain)
Mouse
Control
23.1 + 0.6 (36)
THPO (0.5)
24.1 + 1.0 (12)
THPO (I.0)
23.5 + 0.6 (12)
Ethyl (R)-nipecotate (0.5)
45.5 + 5.4* (12)
Gabaculine (0.5)
53.6 + 1.8" (24)
(0.5) + gabaculine (0.5)
58.8 + 3.6* (12)
Ethyl (R)-nipecotate (0.5) + gabaculine (0.5)
45.5 + 1.5" (12)
Chick (21 days)
Control
24.5 + 1.0 (16)
THPO (i.0)
30.9 + i.i* (16)
Chick (3 days)
Control
25.6 + 0.6 (64)
Hcmo-~-proline (i.0)
24.0 + 1.2 (12)
qis-4-hydromynipecotic acid (i.0)
28.9 ~ 1.5 (15)
(RS)-nipecotic acid (2.0)
24.3 + 0.8 (16)
Ethyl (R)-nipecotate (i.0)
32.3 + 1.8" (14)
THPO (i.0)
44.5 + 1.9, (16)
Gabaculine (0.5)
66.8 + 3.5* (8/16)
THPO (i.0) + gabaculine (0.5)
70.2 + 3.5* (8/16)
The drugs were injected intramuscularlyone hour prior to the intramuscu]ar administration of INH (4.0 mmol/kg). Convulsion times are the mean + S E M for the nt~mber of animals given in parenthesis. Only 8/16 animals convulsed in the last two groups and these means are therefore calculated on n = 8. *Significantly different from controls (P < 0.01). Table 2.
Anticonvulsant action of %~4PO against seizures induced in epileptic chicks by intermittent photic stimulation. Incidence of seizures at various times after injection. lh 2h 4h 22h
Control THPO (i.0)
15/24 5/24*
18/24 5/24*
19/24 8/24*
19/24 16/24
*Significantly different from controls at P < 0.003. ~ESULTS The administration of ~FHPO to mice did not delay the onset of INH-induced seizures, in contrast to the effect of ethyl (R)-nipecotate which brought about a doubling of the time to onset of seizures (Table i). The administration of the GABA-e-oxoglutarate aminotransferase (GABA-T) inhibitor, gabaculine, caused a significant delay in the onset of the INH-induced convulsions, but the concurrent administration of the uptake inhibitors did not enhance the gabaculine anticonvulsant action. In contrast to its lack of effect in mice, ~ brought about a significant delay in the onset of INH-induced seizures in both 3-day-old and 21-day-old chicks, but more so in the younge r chicks (Table i). Again, ethyl (R)-nipecotate produced a significant delay in the seizures, but homo-~-proline, cis-4-hydroxynipecotic acid and (RS)-nipecotic acid
Preliminary Notes
14
itself were without effect in the chicks. Although both gabaculine and THPO produced an anticonvulsant effect in chicks, the action of the oombined drug treatment was only slightly better than that of gabaculine itself. THPO also significantly decreased the number of epileptic chicks having convulsions due to intermittent photic stimulation (Table 2). This effect was relatively longlasting, being still observed 4 hours after the administration of THPO. DISCUSSION While the lack of anticonvulsant activity of THPO in mice may simply indicate that the compound is inactive, an alternate possibility is that it does possess anticonvulsant properties per s__eebut does not penetrate the blood-brain-barrier. Since this barrier in very young chicks is less effective in excluding compounds such as GABA (Wood, 1970), the chick model provides an opportunity to test the anticonvulsant efficacy of GABA analogs which do not penetrate a blood-brain-barrier which has reached a more complete stage of development. Use of this mode] in the present study clearly indicates that THPO does possess anticonvulsant properties (Tables 1 and 2) and points to the lack of effect in mice as being due to permeability probl~ns. Supporting evidence for this conclusion is forthcoming from mouse studies in which THPO does not provide protection frQm maximal electroshock and metrazol-induced seizures (unpublished observations), and from the chick studies in which the anticonvulsant action of THPO decreases with the age of the birds as the blood-brain-barrier develops and becomes more efficient (Table i). The lack of antieonvulsant action of homo~-prollne, cis-4-hydro~ynipecotic acid and (RS)-nipecotic acid in 3-day-01d chicks suggests that even in very young birds the blood-brain-barrier is sufficiently developed to exclude these amino carboxylic acids with potent effects on GABA uptake in vitro, particularly since nipecotic acid is known to possess anticonvulsant activity when it is injected directly into the brain (Horton, Collins, Anlezark and Meldr u~, 1979). Since the anticonvulsant actio~ of gabaculine is probably brought about by increased GABA levels, particularly in the nerve endings (Wood, Russell and Kurylo, 1980), and since the antioonvulsant action of THR9 is probably caused by inhibition of the uptake of GABA from the synaptic cleft into glial cells, it seems reasonable to expect an additive effect when the two compounds are administered concurrently. Unexpectedly, the anticonvulsant action of the drug combination was only slightly better than that of gabaculine alone (Table i). The reason for this phenomenon is uncertain but may be due to the THPO interfering with the uptake of gabaculine into the cells, as was observed previously with gabaculine and the GABA uptake inhibitor, ketamine ~kx)d, Geddes, Tsui and Kurylo, 1982). In conclusion, it would seem that glial-selective irthibitors of GABA uptake are promising candidates as antioonvulsant agents, but that the impermeability of the bloodbrain-barrier to these drugs greatly lessens their usefulness. Further research directed towards the development of a glial-se!ective inhibitor which passes readily into the brain would therefore seem a top priority. A~DGEMENT The authors thank Mr. E. Kurylo for his skilled technical assistance. The work was supported by grants MT-3801 and Mr-5893 from the Medical Research Council of Canada and by grants from the Danish Medical Research Concil and the NOVO Foundation. ;~FE~NCES Akkerman, A. M., De Jongh, D. K. and Ve!dstra, H. (1951). Synthetic oxytocics. I. 3-(Piperidyl-(N)-methyl)-indoles and related compounds. Recl. Tray. Chim. Pays-Bas Belg. 70: 899-916. Crichlow, E.C. and Crawford, R.D. (1974). Epileptiform seizures in domestic fowl II. Intermittent ]ight stimulation and electroencephalogram. Canad. J. Physiol. Pharmacol. 52: 424-429. Frey, H.-H., Popp, C. and Loscher, W. (1979). Influence of inhibitors of the high affinity GABA uptake on seizure thresholds in mice. Neuropharmacol. 18: 581-590. Horton, R.W., Collins, J.F., Anlezark, G.M. and Meldrt~, B.S. (1979). Convulsant and anticonvulsant actions in DBA/2 mice of compounds blocking the reuptake of GABA. Eur. J. Pharmacol. 59: 75-83. Iversen, L.L. and Kelly, J.S. (1975). Uptake and metabolism of 7-aminobutyric acid by neurones and glial cells. Biochsm. Pharmacol. 34: 933-938. Krogsgaard-Larsen, P. and Hjeds, H. (1974). Structural analogues of ?-aminoburyric acid (GABA) of the isoxazole enol-betaine type. Synthesis of 5,6,7,8-tetrahydro-4Hisoxazolo [4,5-d] -azepin-3-ol zwitter ion and 4,5,6,7-tetrahydr isoxazol [4,5-c] pyridin-3-ol zwitterion. Acta Chem. Scand. B28: 533-538. Krogsgaard-Larsen, ~., Thyssen, K. and Schau~m/rg, K. (1978). Inhibitors of GABA uptake. Synthesis and --H NMR spectroscopic investigations of guvacine, (3RS, 4SR)-4-hydroxypiperidin~3-carboxylic acid and related compounds. Acta Chem. Scand. B32: 327-334. Schousboe, A., Larsson, O.M., Hertz, L. and Krogsgaard-Larsen, P. (1981) Heterocyclic GABA
42
Preliminary Notes
analogues as selective inhibitors of astroglial C~%BA uptake. In: Amino Acid Tranmmitters (De Feudis, F.V. and Mandel, P., eds.) pp. 135~i145. Thorbek, P., Hjeds, H. and Schaumburg, K. (1981). Syntheses and ~H h~4R spectroscopic investigations of scrne pyrrolidine carboxylic acids designed as potential glial GABA uptake ir[hibitors. Acta Chem. Scand. B35: 473-479. Wood, J.D. (1970). Seizures induced by hyperbaric oxygen and cerebral ¥-eminobutyric acid in chicks during development. J. Neurochem. 17: 573-579. Wood, J.D., Geddes, J.W., Tsui, S-K. and Kurylo, E. (1982). Combined effects of a metabolic inhibitor (gabaculine) and an uptake inhibitor (ketamine) on the T-aminobutyrate system in mouse brain. J. Neurochem. (in press). Wood, J.D., Russell, M.P. and Kurylo, E. (1980). The Y-aminobutyrate content of nerve endings (synaptosomes) in mice after the injection of Y-aminoburyrate-elevating agents: a possible role ~n anticonvulsant activity. J. Neurochem. 35: 125-130.