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Abnormalities in the levels of extracellular and tissue amino acids in the brain of the seizure-susceptible rat
130
Epilepsy Res., 3 (1989) 130-131
Elsevier ERS 00223
Abnormalities in the levels of extracellular and tissue amino acids in the brain of the seizure-susceptible rat
Anders Le~mann Institute of Neurobiology,
Univer,sity of Gbteborg, P.O. B. 33031, S-400 33 Cdteborg (Sweden)
(Received 2 February 1988; accepted 25 April 1988) Key words: Seizure-susceptible
rat; Amino acids; Brain dialysis; Aspartate; Taurine; Phosphoethanoiamine;
Tyrosine
Basal and high potassium-stimulated release of endogenous amino acids was measured using brain dialysis in the hippocampus of urethane-anesthetized seizure-resistant (SR) and seizure-susceptible (SS) rats. Moreover, the tissue level of amino acids was determined in the hippocampus, sensorimotor cortex, cerebellum and corpus striatum. The basal extracellular concentration of amino acids did not differ between SR and SS rats. However, aspartate release was higher, and taurine and phosphoethanolamine release was lower in SS rats during stimulation with 100 mM K+. Several strain differences were observed with regard to regional tissue levels of amino acids. Aspartate was significantly elevated in the hippocampus, cortex and cerebellum of SS animals, and the catecholamine precursor tyrosine was diminished in all regions examined. Other disparities included a depressed y-aminobutyrate concentration in the hippocampus and cortex, slightly increased levels of phosphoethanolamine in the cerebellum and minor decreases in striatal and cortical taurine. Glutamate, glutamine, serine and alanine concentrations were not significantly altered in any brain area of the SS rat. The results confirm and extend previous findings on abnormalities in aspartate, taurine and phosphoethanolamine regulation in this model. In addition, decreased availability of tyrosine may provide a partial explanation for the well-documented deficiency in cerebral norepinephrine in the SS strain.
INTRODUCTION It is well known that several types of human epilepsy include genetic components’ and this has fostered the development of various animal models of inherited susceptibility to epilepsy. The most widely used rat model is the seizure-susceptible (SS; also referred to as genetically epilepsy-prone) rat of Sprague-Dawley origin**. In contrast to the seizure-resistant (SR) rat, the SS rat exhibits a patCorrespondence to: Anders Lehmann, Institute of Neurobiology. University of Goteborg, P.O.B. 33031, S-400 33 Goteborg, Sweden.
tern of wild-running and clonic-tonic seizures when exposed to high-intensity sound**. The animals were originally designated ‘audiogenic’ and most studies have focused on the auditory system. It may be argued that the significance of the model for human epilepsy is questionable since audiogenie epilepsy is extremely rare in man. However, the term ‘audiogenic’ is misleading as the SS rat has a decreased threshold for most types of epileptogenic stimuli**. Consequently, most of the work carried out recently has been directed at finding anomalies in transmitter systems in different brain regions of these rats, and to relate such changes to the SS state.
0920-1211/89/$03.50 @ 1989 Elsevier Science Publishers B.V. (Biomedical Division)
131 Abnormalities in monoaminergic transmission have been shown to contribute to the sensitivity of the SS rat to epileptogenic challenge13. In view of the ubiquity of synapses utilizing amino acids as transmitters, these compounds may be equally interesting. While initial studies did not disclose any differences between the SR and SS strains in tissue levels of amino acids in different brain regions10,12, Chapman et a1.4 reported multiple disparities in this respect. In addition, Ribak et a1.19observed altered regional levels of neuroactive amino acids in the SS brain, but certain important aspects of their results were not compatible with those of Chapman et a1.4. The present study was initiated to yield further information on this issue. Although altered concentrations of transmitter amino acids in tissue biopsies undoubtedly indicate dysfunction of the systems, such data do not accurately mirror the compartment directly involved in neurotransmission - the extracellular space. This issue has been approached by intracranial dialysis of prelabeled amino acids in the SS rat16. However, prelabeled and endogenous amino acids frequently exhibit differences in their release characteristics. In this investigation, SS and SR rats were compared with respect to basal and high potassium-stimulated release of endogenous amino acids in the hippocampus. METHODS Animals
Male SS rats (‘maximal responders’; ‘GEPR-9’) were provided by Dr. Leena Tuomisto, University of Kuopio, Finland. The stock was originally derived from the Uaz:AGS(SD) colony of the University of Arizona. The seizure susceptibility of the animals was confirmed in the breeder’s laboratory by exposing them to loud noise on 3 separate occasions. Control male Sprague-Dawley SR rats were obtained from ALAB, Sodertalje, Sweden. After their arrival, the rats were kept for 3 months prior to experimentation; at this time they weighed 400-450 g. In total, 12 SS and 13 SR rats were used. Intracranial dialysis
The animals were anesthetized
with 1.25 g/kg
urethane intraperitoneally in combination with atropine sulfate (0.2 mg/kg). Rectal temperature was maintained at 37.5 + 0.5 “C with a heating lamp. The head was fixed horizontally in a Kopf head holder and the calvarium was exposed. A circular trephination was made, the dura was opened with a syringe needle and a dialysis cannula’l was implanted in the right hippocampus 5.0 mm behind the bregma, 5.0 mm lateral to the sagittal suture and 7.5 mm below the cortical surface. The hippocampus was chosen since it is a region with low threshold for epileptiform activity. One SR and 1 SS rat were perfused on each experimental occasion to avoid systematical errors, The perfusates of paired experiments were analyzed on the same occasion. The dialysis tubing was perfused with Krebs-Ringer bicarbonate buffer (prewarmed to 37 “C and saturated with 95% 0,/5% CO,) at 3.5 ,&min for 90 min. The buffer contained (in mM): NaCl 122; KC1 3; MgSO, 1.2; KH,PO, 0.4; NaHCO, 25 and CaCl, 1.2. Perfusion fluid was prepared immediately before the experiment. After an equilibration period of 90 min, three 10 min fractions were collected, separated by intervals of 20 min. The perfusion solution was then exchanged for buffer containing 100 mM potassium chloride with an equimolar reduction of sodium chloride to maintain normal ionic strength. The dialysate was collected just as during the prestimulation period. The samples were frozen immediately at -20 “C and amino acid analysis was performed within 2 weeks. Amino acids were quantified with automatic precolumn derivatization with o-phthalaldehyde and high-performance liquid chromatographic separation with fluorescence detection”. Determination of tissue amino acids
Directly after the dialysis experiment, the rats were decapitated and their heads were immersed in liquid nitrogen. The head was repeatedly cooled in liquid nitrogen while the brain was removed. Brains were stored at -80 “C, and the hippocampus, the sensorimotor cortex, the corpus striatum and the cerebellum from all animals were isolated on the same occasion”. Dissection was carried out at an ambient temperature of 3 “C and the brains were kept cool with liquid nitrogen. The cerebellar
132
sample included the vermis and the lateral hemispheres and the temporal part of the left (non-dialyzed) hippocampus was taken. The tissue was weighed and homogenized in ice-cold 0.6 M perchloric acid. The homogenate was centrifuged at 10,000 x g for 10 min and the supernatant was neutralized with 0.6 M NaOH. The extracts were frozen and analyzed within 2 weeks.
2no
RESULTS Extracellular (perfusate) amino acids The concentrations of different amino acids were close to steady-state levels during perfusion with low potassium medium (Fig. 1). The mean levels of the 3 prestimulation samples were (SR vs. SS): aspartate 0.11 + 0.01 vs. 0.13 + O.OlpM; glutamate 0.33 + 0.04 vs. 0.38 -+ 0.03,uM; glutamine 26.8 f 2.6 vs. 24.4 f 2.6 FM; y-aminobutyrate (GABA) 27.9 * 8.1 vs. 16.9 F 2.9 nM; taurine 2.63 f 0.24 vs. 2.35 10.17pM; phosphoethanolamine (PEA) 0.69 + 0.06 vs. 0.63 + 0.05 PM; tyrosine 0.58 It 0.06 vs. 0.44 It 0.05 ,uM and alanine 1.44 10.25 vs. 1.58 + 0.26pM. None of these differences was statistically significant with Student’s t test. Stimulation with high levels of potassium induced increases in aspartate, glutamate, GABA (Fig. l), taurine and PEA (Fig. 2). The elevation of GABA was, relative to baseline, most striking. The peak of taurine and PEA release was delayed compared to aspartate and glutamate. Alanine was not affected by high potassium (Fig. 3), but both glutamine (Fig. 2) and tyrosine (Fig. 3) decreased significantly, Intergroup comparisons revealed a significantly (P < 0.05) higher release of aspartate in the last sample collected from SS rats and lower levels of taurine and PEA in 2 out of 3 fractions. The stimulated release of glutamate was
Fig. 1. Basal and high potassium-stimulated release of aspartate, glutamate and GABA from the hippocampus of SR (solid bars) and SS (hatched bars) rats. The first 3 fractions were collected during perfusion with buffer containing 3.4 mM potassium and the last 3 fractions were sampled during perfusion with 100 mM potassium. Bars represent mean t- S.E.M.; star denotes significant difference between SR and SS at P < 0.05 (Student’s unpaired f test).
Time (min) hih potassium
I
L
f
2.0
3 ?i
ss $j!t t
2.0
!i
G
0.0 O-10
50.40
00-10
Time
O-10
*o-40
00-70
00.100
(mi”)
to-loo
Time (mln)
*PO-130
too-100
high potasswm
tao-rto
100-100
133
T
I
T
high potawum
I
Time (min)
I
?o
1
hiih potassium
I
T
__
Time (min) high potassium I
I
IT
hiih ptassium
I
,
Fig. 3. Release of tyrosine and alanine in SR and SS rats. Conditions are described in Fig. 1. Tyrosine was significantly reduced in SR and SS animals during perfusion with high potassium (P < 0.05; Student’s paired t test) with the exception of the first high potassium sample from SR rats.
initially higher in the SS rats, although this could not be verified statistically. GABA release tended to be lower in SS rats, but the differences were not statistically significant.
Fig. 2. Release of taurine, phosphoethanolamine and glutamine in SR and SS rats. Conditions are described in Fig. 1. One star: P < 0.05; two stars: P < 0.01 (SR vs. SS). Glutamine was decreased significantly in both groups during high potassium stimulation (P < 0.05, Student’s paired t test).
134 Regional tissue content of amino acids
DISCUSSION
Of the neuroactive amino acids, aspartate levels showed the most consistent changes in brains from SS rats. This amino acid was significantly higher in the hippocampus, cortex and cerebellum from SS rats (Table I). GABA was depressed in the hippocampus and cortex, and taurine was moderately reduced in the striatum and cortex. PEA was slightly higher in the cerebellum. There were no differences between the strains with regard to glutamate, glutamine, serine or alanine. However, tyrosine was significantly lower in all regions from SS rats. In Table II, the present observations are summarized and compared with previous findings.
Evidence has accumulated that dysfunction of amino acid-mediated neurotransmission occurs in the SS rat. GABA-mediated inhibition is decreased in the inferior colliculus of the SS animals. The density of GABA-containing neurons is increased20 which possibly reflects compensatory mechanisms. Using prelabeling with radioactive amino acids in vivo, Lehmann et a1.16found an elevated spontaneous and potassium-stimulated release of D-[3H]aspartate, whereas the release of GABA, taurine, norepinephrine and its metabolites was normal in the hippocampus16. This find-
TABLE I Regional levels of amino aciak (pmolfg wet weight) in brains from seizure-resrjtcmt (SR) and seizure-susceptible (SS) rats
Gitu
ASP
SR
Hippocampus Cortex Cerebellum Striatum
ss
11.16 rt 11.96 rt 10.83 + 10.98 k
0.49 0.50 0.26 0.23
SR
11.55 + 11.92 + 11.18 + 10.55 k
0.54 0.26 0.31 0.38
GABA
Hippocampus Cortex Cerebellum Striatum
1.60 4 0.07 1.42 i: 0.05 2.93 + 0.29
2.11 + 0.15** 1.34 zk0.0.5** 1.45 2 0.05 2.72 + 0.22 -__
Hippocampus Cortex Cerebellum Striatum
Tyr --_ 0.14 0.13 0.14 0.12
0.08 0.13 0.05 0.08
1.80 + 3.01 + 2.06 t 1.60 t
O.lO* 0.16** O.lO*** 0.06
4.05 2.33 7.01 1.9s
0.19 0.08 0.41 0.13
3.63 + 2.35 f 6.27 + 1.87 f
0.11 0.13 0.25 0.11
+ + + +
PEA
Tau
6.30 6.72 5.58 8.91
1.52 + 2.37 + 1.61 + 1.44 f Gh
2.70 f: 0.15
Hippocampus Cortex Cerebellum Striatum
ss
+ 0.21 zk0.17 i: 0.14 F 0.19
6.43 6.31 5.23 7.99
+ + + t
0.18 0.12* 0.22 0.25**
1.61 + 0.05
1.42 + 0.07
1.47 -I 0.07
0.59 + 0.01
0.63 k 0.02*
2.21 t 0.07
2.07 + 0.12
Ala
zk0.01 + 0.00 t 0.01 + 0.01
0.12 0.10 0.11 0.08 -
zk0.01* + o.oO*** t o.oO** k 0.01***
0.76 5 0.04 0.54 + 0.03 0.46 rfI0.03 0.55 t 0.03
Ser
Hippocampus Cortex Cerebellum
_..-______-
1.64 + 0.06
1.16 k 0.93 It 0.71 It 0.92 ;t
0.06 0.04 0.04 0.05
1.17 t 0.92 + 0.79 t 0.87 i-
0.08 0.04 0.06 0.04
Results are mean + SEM; *P < 0.05; **P < 0.01; ***P < 0.001 (Student’s unpaired ttest).
-
0.81 + 0.06 0.54 f 0.03 0.45 + 0.03 0.55 * 0.05
135 TABLE II Changes in cerebral amino acid levels in the SS rat expressed GlU Reference
Cortex Hippocampus Striatum Inferior colliculus Cerebellum
12
GABA
Asp 4
19
Pw
12
4
19
Pw
12
4
19
107
94
ND ND
73* 82*
160 ND ND
100 103 96
103 ND ND
97 76 67*
111 ND ND
127’ 118* 111
91 ND ND
125 141* 107
94 ND ND
105 ND
86 91
190* 122
ND 103
114 ND
68’ 78
115 71
ND 128:
112 ND
135 106
230’ 73
pw
84* 78* 93 ND 102
Gln
Tau
Cortex Hippocampus Striatum Inferior colliculus Cerebellum
aspercentof SR control: comparison between present and previous findings
102 ND ND
104 94 86*
147* ND ND
94 102 90’
101 ND ND
86 57’ 63*
144 ND ND
101 90 96
110 ND
110 90
242* 117
ND 94
107 ND
67* 53*
80 104
ND 89
The cortical samples were taken from the sensorimotor area (refs. 12,19 and present work) or from unspecified areas (ref. 4). The collicular sample included the entire region (refs. 12 and 4) or the central nucleus only (ref. 19). ND = not determined; asterisk indicates P < 0.05, pw = present work.
ing is contrasted by the report of Chapman et a1.4 who did not detect differences in the release of D[3H]aspartate from hippocampal slices from SR and SS rats. The present study shows that both basal and potassium-evoked efflux of glutamate were similar in the 2 strains. However, stimulated release of endogenous aspartate was higher in the epileptic rats but only 60-70 min after high K+ perfusion began. Thus, the rapid response of aspartate to Kf stimulation does not differ between SR and SS rats, but the decline is slower in the epileptic animals. The influence of urethane anesthesia on amino acid release is not known, but it should be equal in both strains. D-Aspartate is taken up both in putative glutamatergic and aspartatergic neurons (as well as in glial cells), and since the former population is probably quantitatively more important, glutamatergic nerve cells may be responsible for most of the release of pre-labeled D-aspartate. It was consequently expected that SS rats would release more endogenous glutamate and aspartate. The discrepant results may be due to differences in cellular origin (i.e., neurons vs. astrocytes) with respect to releasable pools of endogenous and pre-labeled amino acids. The results indicate that aspartate, an excitatory and epilepto-
genie amino acid, is a determinant for seizure susceptibility. The efficiency of D-Zamino-7-phosphonoheptanoate, an excitatory amino acid receptor antagonist, as an anticonvulsant in the SS rat supports this notion’. Under basal conditions, extracellular taurine and PEA did not differ between the strains. In contrast, SS rats released smaller amounts of these amino acids after stimulation with a high concentration of potassium. A close correlation between extracellular taurine and PEA has been noted previously”. The total tissue level of taurine and PEA is similar in the hippocampus (present results) and whole brain3 of SS and SR rats. However, subcellular fractionation of whole brains discloses a selective depression of taurine and PEA in the P,B (synaptosomal) fraction from SS rats3. The reduction of stimulated release may therefore reflect the selective deficiency in nerve terminal pools of the amino acids. Such a relationship does not seem to exist for aspartate, since there is no difference between SS and SR animals with respect to the synaptosomal content of this amino acid3. The global decrease in taurine transport in the SS rat* may also contribute to the reduced release. It is presently not known how the anomalies in taurine and
136
PEA release relate to seizure susceptibility. Taurine is antiepileptic in the SS rat”, and it is possible that the depression of evoked release of the amino acid in part accounts for the low seizure threshold. Extracellular glutamine and tyrosine were suppressed to a similar extent in the 2 groups by depolarization with high potassium. Inasmuch as glutamine is the precursor of glutamate and GABA, and tyrosine is the precursor of catecholamines, the response may mirror increased demand for the amino acids to compensate for the enhanced turnover of their products. Selective diminution of glutamine and tyrosine after high potassium stimulation in vivo has previously been reported to occur in the rat striatum’. The regional tissue concentration (which approximates intracellular levels) of GABA, taurine and PEA exhibited some differences between the rat strains. The most consistent difference was a significant elevation of aspartate in all regions except the striatum, and deficiencies in tyrosine in all areas. Tissue aspartate is probably localized in both metabolic and transmitter pools, and there is at present no method for estimating these separately. It is thus unknown if the increased content of aspartate derives from one or both pools. However, the generalized increase in this excitant may sensitize the SS rat to epileptogenic stimuli. The extracellular concentration of a neuroactive amino acid is more important than the intracellular level from the neurotransmission point of view. The finding that hippocampal levels of intracellular and stimulated extracellular aspartate are elevated in parallel in the SS strain may be coincidental. No such relationship was seen for taurine and PEA. Furthermore, neither basal nor evoked release of amino acids reflect intracellular concentrations23, and this lack of correlation is also observed during drug-induced seizures”. The SS rat has an abnormally low tissue level of norepinephrine in many brain areas5,14. The underlying biochemical factors responsible for the suppression of norepinephrine have not been identified. They can neither be ascribed to alterations in the activity of the rate-limiting anabolic enzyme tyrosine hydroxylase5, nor to changes in pyridoxal phosphate6, an important regulator of the enzyme. The finding that the norepinephrine precursor ty-
rosine is lower in all regions examined may provide an explanation for the noradrenergic deficiency. While this suggestion warrants further investigation, it should be noted that cerebellar tyrosine is decreased, but norepinephrine is normal’ or eievated14. Huxtable and Laird’” and Huxtable et al.” reported that amino acid concentrations are indistinguishable in SS and SR brains. In contrast, Chapman et al.4 and Ribak et al.” found multiple differences between many amino acids in several areas. However, the results of the latter authors were not totally in agreement, and the present findings show little overlap with previous observations (summarized in Table II). It is indeed unexpected that the results of different laboratories would differ so markedly considering the rather straightforward methodology employed. One possibility is that although the animals originally derive from the same colony, genetic drift may have occurred. Methodological differences are, however, more probably responsible for the discrepancies. The urethane anesthesia used in the present experiments may have affected amino acid levels, and the higher levels of aspartate in SS rats may be secondary to mild hypoglycemia. However, this caveat may not be important, since the amino acid levels reported here are within the normal range. Furthermore, it is unlikely that cerebral amino acids of SS and SR rats would be differently affected by urethane. Age and/or sex differences between animals used in this and previous studies may be of some significance. In conclusion, the supersensitivity of aspartate release after prolonged potassium stimulation, combined with depressed taurine release, may constitute a basis for the low seizure threshold in the SS rat. Furthermore, reduced tissue levels of tyrosine may be partly responsible for the previously reported deficiency in norepinephrine. While it is now evident that the SS rat displays multiple abnormalities in regional amino acid levels, no uniform picture can be obtained when previous and present data are compared. ACKNOWLEDGEMENTS I wish to express my sincere gratitude
to Dr.
137
Leena Tuomisto for making the SS rats available, and to Ms. Birgitta Karlsson for technical assistance. This study was supported by the Ahlen, Ericsson, Wiberg, Swedish Society for Medical Research and Hierta-Retzius Foundations. The support of Dr. Anders Hamberger is gratefully acknowledged (MRC Grant No. 12X-00164).
REFERENCES 1 Andersen, V.E., Genetics in the epilepsies, Trends Neurosci., 8 (1985) 513-516. 2 Bonhaus, D.W. and Huxtable, R.J., Seizure-susceptibility and decreased taurine transport in the genetically epileptic rat, Neurochem. Int., 6 (1984) 365-368. 3 Bonhaus, D. W., Lippincott, SE. and Huxtable, R. J., Subcellular distribution of neuroactive amino acids in brains of genetically epileptic rats, Epilepsia, 25 (1984) 564-568. 4 Chapman, A., Faingold, C.L., Hart, G.P., Bowker, H.M. and Meldrum, B.S., Brain regional amino acid levels in seizure susceptible rats: changes related to sound-induced seizures, Neurochem. Int., 8 (1986) 273-279. 5 Dailey, J.W. and Jobe, P.C., Indices of noradrenergic function in the central nervous system of seizure-naive genetically epilepsy-prone rats, Epilepsia, 27 (1986) 665-670. 6 Ebadi, M., Jobe, P.C. and Laird, II, H.E., The status of vitamin B, metabolism in brains of genetically epilepsy-prone rats, Epilepsia, 26 (1985) 353-359. 7 Faingold, C.L., Meldrum, B.S. and Millan, M.H., Blockade of audiogenic seizure susceptibility by focal injection into the inferior colliculus of an excitant amino acid antagonist, Rr. J. Pharmacol., 84 (1984) 95P. 8 Faingold, CL., Gehlbach, G. and Caspary, D.M., Decreased effectiveness of GABA-mediated inhibition in the inferior colliculus of the genetically epilepsy-prone rat, Exp. Neural., 93 (1986) 145-159. 9 Girault, J.A., Barbeito, L., Spampinato, U., Gozlan, H., Glowinsky, J. and Besson, M.-J., In vivo release of endogenous amino acids from the rat striatum: further evidence for a role of glutamate and aspartate in corticostriatal neurotransmission, J. Neurochem., 47 (1986) 98- 106. 10 Huxtable, R.J. and Laird, II, H.E., Are amino acid patterns necessarily abnormal in epileptic brains? Studies on the genetically seizure-susceptible rat, Neurosci. Lett., 10 (1978) 341-345. 11 Huxtable, R.J. and Laird, H.E., The prolonged anticonvulsant action of taurine on genetically determined seizuresusceptibility, Can. J. Neural. Sci., 5 (1978) 215-221. 12 Huxtable, R.J., Laird, H.E., Bonhaus, D. and Thies, A.C., Correlations between amino acid concentrations in brains of seizure susceptible and seizure resistant rats, Neurochem. Int., 4 (1982) 73-78.
NOTE ADDED IN PROOF I have recently found that extracellular levels of taurine and PEA are particularly sensitive to changes in osmolality. It is therefore possible that the lower Kf-stimulated release of these amino acids in the SS rat hippocampus reflects derangements in osmoregulation.
13 Jobe, P.C., Picchioni, A.L. and Chin, L., Role of brain norepinephrine in audiogenic seizure in the rat, J. Pharmacol. Exp. Ther., 184 (1973) l-10. 14 Jobe, P.C., Laird, II, H.E., Ko, K.H., Ray, T. and Dailey, J.W., Abnormalities in monoamine levels in the central nervous system of the genetically epilepsy-prone rat, Epilepsia, 23 (1982) 359-366. 15 Lehmann, A. and Hamberger,
A., A possible interrelationship between extracellular taurine and phosphoethanolamine in the hippocampus, J. Neurochem., 42 (1984)
1286-1290. 16 Lehmann, A., Sandberg,
M. and Huxtable, R.J., In vivo release of neuroactive amines and amino acids from the hippocampus of seizure-resistant and seizure-susceptible rats, Neurochem.
17 Lehmann,
Int., 8 (1986) 513-520.
A., Alterations in hippocampal extracellular amino acids and purine catabolites during limbic seizures induced by folate injections into the rabbit amygdala, Neuroscience, 22 (1987) 573-578. 18 Lindroth, P., Sandberg, M. and Hamberger, A., Liquid chromatographic determination of amino acids after precolumn fluorescence derivatization. In: A.A. Boulton, G.B. Baker and J.D. Wood (Eds.), Neuromethods: Amino Acids, Vol. 3, Humana Press, Clifton, NJ, 1985, pp. 97-116. 19 Ribak, C.E., Byun, M.Y., Ruiz, G.T. and Reiffenstein, R.J., Increased levels of amino acid neurotransmitters in the inferior colliculus of the genetically epilepsy-prone rat, Epilepsy Res., 2 (1988) 9-13. 20 Roberts, R.C. and Ribak, C.E., Anatomical changes of the GABAergic system in the inferior colliculus of the genetically epilepsy-prone rat, Life Sci., 39 (1986) 789-798. 21 Sandberg, M., Butcher, S.P. and Hagberg, H., Extracellular overflow of neuroactive amino acids during severe insulin-induced hypoglycemia - in vivo dialysis of the rat hippocampus,J. Neurochem., 47 (1986) 178-184. 22 Tacke, U., Audiogenic Seizures in Rats - an Experimental Study of a Genetic Model of Epilepsy, Ph.D. Thesis, University of Kuopio, 1984. 23 Tossman, U., Jonsson, G. and Ungerstedt, U., Regional distribution and extracellular levels of amino acids in rat central nervous system, Acta Physiol. Stand., 127 (1986) 533-545.
Epilepsy Res., 3 (1989) 130-131
Elsevier ERS 00223
Abnormalities in the levels of extracellular and tissue amino acids in the brain of the seizure-susceptible rat
Anders Le~mann Institute of Neurobiology,
Univer,sity of Gbteborg, P.O. B. 33031, S-400 33 Cdteborg (Sweden)
(Received 2 February 1988; accepted 25 April 1988) Key words: Seizure-susceptible
rat; Amino acids; Brain dialysis; Aspartate; Taurine; Phosphoethanoiamine;
Tyrosine
Basal and high potassium-stimulated release of endogenous amino acids was measured using brain dialysis in the hippocampus of urethane-anesthetized seizure-resistant (SR) and seizure-susceptible (SS) rats. Moreover, the tissue level of amino acids was determined in the hippocampus, sensorimotor cortex, cerebellum and corpus striatum. The basal extracellular concentration of amino acids did not differ between SR and SS rats. However, aspartate release was higher, and taurine and phosphoethanolamine release was lower in SS rats during stimulation with 100 mM K+. Several strain differences were observed with regard to regional tissue levels of amino acids. Aspartate was significantly elevated in the hippocampus, cortex and cerebellum of SS animals, and the catecholamine precursor tyrosine was diminished in all regions examined. Other disparities included a depressed y-aminobutyrate concentration in the hippocampus and cortex, slightly increased levels of phosphoethanolamine in the cerebellum and minor decreases in striatal and cortical taurine. Glutamate, glutamine, serine and alanine concentrations were not significantly altered in any brain area of the SS rat. The results confirm and extend previous findings on abnormalities in aspartate, taurine and phosphoethanolamine regulation in this model. In addition, decreased availability of tyrosine may provide a partial explanation for the well-documented deficiency in cerebral norepinephrine in the SS strain.
INTRODUCTION It is well known that several types of human epilepsy include genetic components’ and this has fostered the development of various animal models of inherited susceptibility to epilepsy. The most widely used rat model is the seizure-susceptible (SS; also referred to as genetically epilepsy-prone) rat of Sprague-Dawley origin**. In contrast to the seizure-resistant (SR) rat, the SS rat exhibits a patCorrespondence to: Anders Lehmann, Institute of Neurobiology. University of Goteborg, P.O.B. 33031, S-400 33 Goteborg, Sweden.
tern of wild-running and clonic-tonic seizures when exposed to high-intensity sound**. The animals were originally designated ‘audiogenic’ and most studies have focused on the auditory system. It may be argued that the significance of the model for human epilepsy is questionable since audiogenie epilepsy is extremely rare in man. However, the term ‘audiogenic’ is misleading as the SS rat has a decreased threshold for most types of epileptogenic stimuli**. Consequently, most of the work carried out recently has been directed at finding anomalies in transmitter systems in different brain regions of these rats, and to relate such changes to the SS state.
0920-1211/89/$03.50 @ 1989 Elsevier Science Publishers B.V. (Biomedical Division)
131 Abnormalities in monoaminergic transmission have been shown to contribute to the sensitivity of the SS rat to epileptogenic challenge13. In view of the ubiquity of synapses utilizing amino acids as transmitters, these compounds may be equally interesting. While initial studies did not disclose any differences between the SR and SS strains in tissue levels of amino acids in different brain regions10,12, Chapman et a1.4 reported multiple disparities in this respect. In addition, Ribak et a1.19observed altered regional levels of neuroactive amino acids in the SS brain, but certain important aspects of their results were not compatible with those of Chapman et a1.4. The present study was initiated to yield further information on this issue. Although altered concentrations of transmitter amino acids in tissue biopsies undoubtedly indicate dysfunction of the systems, such data do not accurately mirror the compartment directly involved in neurotransmission - the extracellular space. This issue has been approached by intracranial dialysis of prelabeled amino acids in the SS rat16. However, prelabeled and endogenous amino acids frequently exhibit differences in their release characteristics. In this investigation, SS and SR rats were compared with respect to basal and high potassium-stimulated release of endogenous amino acids in the hippocampus. METHODS Animals
Male SS rats (‘maximal responders’; ‘GEPR-9’) were provided by Dr. Leena Tuomisto, University of Kuopio, Finland. The stock was originally derived from the Uaz:AGS(SD) colony of the University of Arizona. The seizure susceptibility of the animals was confirmed in the breeder’s laboratory by exposing them to loud noise on 3 separate occasions. Control male Sprague-Dawley SR rats were obtained from ALAB, Sodertalje, Sweden. After their arrival, the rats were kept for 3 months prior to experimentation; at this time they weighed 400-450 g. In total, 12 SS and 13 SR rats were used. Intracranial dialysis
The animals were anesthetized
with 1.25 g/kg
urethane intraperitoneally in combination with atropine sulfate (0.2 mg/kg). Rectal temperature was maintained at 37.5 + 0.5 “C with a heating lamp. The head was fixed horizontally in a Kopf head holder and the calvarium was exposed. A circular trephination was made, the dura was opened with a syringe needle and a dialysis cannula’l was implanted in the right hippocampus 5.0 mm behind the bregma, 5.0 mm lateral to the sagittal suture and 7.5 mm below the cortical surface. The hippocampus was chosen since it is a region with low threshold for epileptiform activity. One SR and 1 SS rat were perfused on each experimental occasion to avoid systematical errors, The perfusates of paired experiments were analyzed on the same occasion. The dialysis tubing was perfused with Krebs-Ringer bicarbonate buffer (prewarmed to 37 “C and saturated with 95% 0,/5% CO,) at 3.5 ,&min for 90 min. The buffer contained (in mM): NaCl 122; KC1 3; MgSO, 1.2; KH,PO, 0.4; NaHCO, 25 and CaCl, 1.2. Perfusion fluid was prepared immediately before the experiment. After an equilibration period of 90 min, three 10 min fractions were collected, separated by intervals of 20 min. The perfusion solution was then exchanged for buffer containing 100 mM potassium chloride with an equimolar reduction of sodium chloride to maintain normal ionic strength. The dialysate was collected just as during the prestimulation period. The samples were frozen immediately at -20 “C and amino acid analysis was performed within 2 weeks. Amino acids were quantified with automatic precolumn derivatization with o-phthalaldehyde and high-performance liquid chromatographic separation with fluorescence detection”. Determination of tissue amino acids
Directly after the dialysis experiment, the rats were decapitated and their heads were immersed in liquid nitrogen. The head was repeatedly cooled in liquid nitrogen while the brain was removed. Brains were stored at -80 “C, and the hippocampus, the sensorimotor cortex, the corpus striatum and the cerebellum from all animals were isolated on the same occasion”. Dissection was carried out at an ambient temperature of 3 “C and the brains were kept cool with liquid nitrogen. The cerebellar
132
sample included the vermis and the lateral hemispheres and the temporal part of the left (non-dialyzed) hippocampus was taken. The tissue was weighed and homogenized in ice-cold 0.6 M perchloric acid. The homogenate was centrifuged at 10,000 x g for 10 min and the supernatant was neutralized with 0.6 M NaOH. The extracts were frozen and analyzed within 2 weeks.
2no
RESULTS Extracellular (perfusate) amino acids The concentrations of different amino acids were close to steady-state levels during perfusion with low potassium medium (Fig. 1). The mean levels of the 3 prestimulation samples were (SR vs. SS): aspartate 0.11 + 0.01 vs. 0.13 + O.OlpM; glutamate 0.33 + 0.04 vs. 0.38 -+ 0.03,uM; glutamine 26.8 f 2.6 vs. 24.4 f 2.6 FM; y-aminobutyrate (GABA) 27.9 * 8.1 vs. 16.9 F 2.9 nM; taurine 2.63 f 0.24 vs. 2.35 10.17pM; phosphoethanolamine (PEA) 0.69 + 0.06 vs. 0.63 + 0.05 PM; tyrosine 0.58 It 0.06 vs. 0.44 It 0.05 ,uM and alanine 1.44 10.25 vs. 1.58 + 0.26pM. None of these differences was statistically significant with Student’s t test. Stimulation with high levels of potassium induced increases in aspartate, glutamate, GABA (Fig. l), taurine and PEA (Fig. 2). The elevation of GABA was, relative to baseline, most striking. The peak of taurine and PEA release was delayed compared to aspartate and glutamate. Alanine was not affected by high potassium (Fig. 3), but both glutamine (Fig. 2) and tyrosine (Fig. 3) decreased significantly, Intergroup comparisons revealed a significantly (P < 0.05) higher release of aspartate in the last sample collected from SS rats and lower levels of taurine and PEA in 2 out of 3 fractions. The stimulated release of glutamate was
Fig. 1. Basal and high potassium-stimulated release of aspartate, glutamate and GABA from the hippocampus of SR (solid bars) and SS (hatched bars) rats. The first 3 fractions were collected during perfusion with buffer containing 3.4 mM potassium and the last 3 fractions were sampled during perfusion with 100 mM potassium. Bars represent mean t- S.E.M.; star denotes significant difference between SR and SS at P < 0.05 (Student’s unpaired f test).
Time (min) hih potassium
I
L
f
2.0
3 ?i
ss $j!t t
2.0
!i
G
0.0 O-10
50.40
00-10
Time
O-10
*o-40
00-70
00.100
(mi”)
to-loo
Time (mln)
*PO-130
too-100
high potasswm
tao-rto
100-100
133
T
I
T
high potawum
I
Time (min)
I
?o
1
hiih potassium
I
T
__
Time (min) high potassium I
I
IT
hiih ptassium
I
,
Fig. 3. Release of tyrosine and alanine in SR and SS rats. Conditions are described in Fig. 1. Tyrosine was significantly reduced in SR and SS animals during perfusion with high potassium (P < 0.05; Student’s paired t test) with the exception of the first high potassium sample from SR rats.
initially higher in the SS rats, although this could not be verified statistically. GABA release tended to be lower in SS rats, but the differences were not statistically significant.
Fig. 2. Release of taurine, phosphoethanolamine and glutamine in SR and SS rats. Conditions are described in Fig. 1. One star: P < 0.05; two stars: P < 0.01 (SR vs. SS). Glutamine was decreased significantly in both groups during high potassium stimulation (P < 0.05, Student’s paired t test).
134 Regional tissue content of amino acids
DISCUSSION
Of the neuroactive amino acids, aspartate levels showed the most consistent changes in brains from SS rats. This amino acid was significantly higher in the hippocampus, cortex and cerebellum from SS rats (Table I). GABA was depressed in the hippocampus and cortex, and taurine was moderately reduced in the striatum and cortex. PEA was slightly higher in the cerebellum. There were no differences between the strains with regard to glutamate, glutamine, serine or alanine. However, tyrosine was significantly lower in all regions from SS rats. In Table II, the present observations are summarized and compared with previous findings.
Evidence has accumulated that dysfunction of amino acid-mediated neurotransmission occurs in the SS rat. GABA-mediated inhibition is decreased in the inferior colliculus of the SS animals. The density of GABA-containing neurons is increased20 which possibly reflects compensatory mechanisms. Using prelabeling with radioactive amino acids in vivo, Lehmann et a1.16found an elevated spontaneous and potassium-stimulated release of D-[3H]aspartate, whereas the release of GABA, taurine, norepinephrine and its metabolites was normal in the hippocampus16. This find-
TABLE I Regional levels of amino aciak (pmolfg wet weight) in brains from seizure-resrjtcmt (SR) and seizure-susceptible (SS) rats
Gitu
ASP
SR
Hippocampus Cortex Cerebellum Striatum
ss
11.16 rt 11.96 rt 10.83 + 10.98 k
0.49 0.50 0.26 0.23
SR
11.55 + 11.92 + 11.18 + 10.55 k
0.54 0.26 0.31 0.38
GABA
Hippocampus Cortex Cerebellum Striatum
1.60 4 0.07 1.42 i: 0.05 2.93 + 0.29
2.11 + 0.15** 1.34 zk0.0.5** 1.45 2 0.05 2.72 + 0.22 -__
Hippocampus Cortex Cerebellum Striatum
Tyr --_ 0.14 0.13 0.14 0.12
0.08 0.13 0.05 0.08
1.80 + 3.01 + 2.06 t 1.60 t
O.lO* 0.16** O.lO*** 0.06
4.05 2.33 7.01 1.9s
0.19 0.08 0.41 0.13
3.63 + 2.35 f 6.27 + 1.87 f
0.11 0.13 0.25 0.11
+ + + +
PEA
Tau
6.30 6.72 5.58 8.91
1.52 + 2.37 + 1.61 + 1.44 f Gh
2.70 f: 0.15
Hippocampus Cortex Cerebellum Striatum
ss
+ 0.21 zk0.17 i: 0.14 F 0.19
6.43 6.31 5.23 7.99
+ + + t
0.18 0.12* 0.22 0.25**
1.61 + 0.05
1.42 + 0.07
1.47 -I 0.07
0.59 + 0.01
0.63 k 0.02*
2.21 t 0.07
2.07 + 0.12
Ala
zk0.01 + 0.00 t 0.01 + 0.01
0.12 0.10 0.11 0.08 -
zk0.01* + o.oO*** t o.oO** k 0.01***
0.76 5 0.04 0.54 + 0.03 0.46 rfI0.03 0.55 t 0.03
Ser
Hippocampus Cortex Cerebellum
_..-______-
1.64 + 0.06
1.16 k 0.93 It 0.71 It 0.92 ;t
0.06 0.04 0.04 0.05
1.17 t 0.92 + 0.79 t 0.87 i-
0.08 0.04 0.06 0.04
Results are mean + SEM; *P < 0.05; **P < 0.01; ***P < 0.001 (Student’s unpaired ttest).
-
0.81 + 0.06 0.54 f 0.03 0.45 + 0.03 0.55 * 0.05
135 TABLE II Changes in cerebral amino acid levels in the SS rat expressed GlU Reference
Cortex Hippocampus Striatum Inferior colliculus Cerebellum
12
GABA
Asp 4
19
Pw
12
4
19
Pw
12
4
19
107
94
ND ND
73* 82*
160 ND ND
100 103 96
103 ND ND
97 76 67*
111 ND ND
127’ 118* 111
91 ND ND
125 141* 107
94 ND ND
105 ND
86 91
190* 122
ND 103
114 ND
68’ 78
115 71
ND 128:
112 ND
135 106
230’ 73
pw
84* 78* 93 ND 102
Gln
Tau
Cortex Hippocampus Striatum Inferior colliculus Cerebellum
aspercentof SR control: comparison between present and previous findings
102 ND ND
104 94 86*
147* ND ND
94 102 90’
101 ND ND
86 57’ 63*
144 ND ND
101 90 96
110 ND
110 90
242* 117
ND 94
107 ND
67* 53*
80 104
ND 89
The cortical samples were taken from the sensorimotor area (refs. 12,19 and present work) or from unspecified areas (ref. 4). The collicular sample included the entire region (refs. 12 and 4) or the central nucleus only (ref. 19). ND = not determined; asterisk indicates P < 0.05, pw = present work.
ing is contrasted by the report of Chapman et a1.4 who did not detect differences in the release of D[3H]aspartate from hippocampal slices from SR and SS rats. The present study shows that both basal and potassium-evoked efflux of glutamate were similar in the 2 strains. However, stimulated release of endogenous aspartate was higher in the epileptic rats but only 60-70 min after high K+ perfusion began. Thus, the rapid response of aspartate to Kf stimulation does not differ between SR and SS rats, but the decline is slower in the epileptic animals. The influence of urethane anesthesia on amino acid release is not known, but it should be equal in both strains. D-Aspartate is taken up both in putative glutamatergic and aspartatergic neurons (as well as in glial cells), and since the former population is probably quantitatively more important, glutamatergic nerve cells may be responsible for most of the release of pre-labeled D-aspartate. It was consequently expected that SS rats would release more endogenous glutamate and aspartate. The discrepant results may be due to differences in cellular origin (i.e., neurons vs. astrocytes) with respect to releasable pools of endogenous and pre-labeled amino acids. The results indicate that aspartate, an excitatory and epilepto-
genie amino acid, is a determinant for seizure susceptibility. The efficiency of D-Zamino-7-phosphonoheptanoate, an excitatory amino acid receptor antagonist, as an anticonvulsant in the SS rat supports this notion’. Under basal conditions, extracellular taurine and PEA did not differ between the strains. In contrast, SS rats released smaller amounts of these amino acids after stimulation with a high concentration of potassium. A close correlation between extracellular taurine and PEA has been noted previously”. The total tissue level of taurine and PEA is similar in the hippocampus (present results) and whole brain3 of SS and SR rats. However, subcellular fractionation of whole brains discloses a selective depression of taurine and PEA in the P,B (synaptosomal) fraction from SS rats3. The reduction of stimulated release may therefore reflect the selective deficiency in nerve terminal pools of the amino acids. Such a relationship does not seem to exist for aspartate, since there is no difference between SS and SR animals with respect to the synaptosomal content of this amino acid3. The global decrease in taurine transport in the SS rat* may also contribute to the reduced release. It is presently not known how the anomalies in taurine and
136
PEA release relate to seizure susceptibility. Taurine is antiepileptic in the SS rat”, and it is possible that the depression of evoked release of the amino acid in part accounts for the low seizure threshold. Extracellular glutamine and tyrosine were suppressed to a similar extent in the 2 groups by depolarization with high potassium. Inasmuch as glutamine is the precursor of glutamate and GABA, and tyrosine is the precursor of catecholamines, the response may mirror increased demand for the amino acids to compensate for the enhanced turnover of their products. Selective diminution of glutamine and tyrosine after high potassium stimulation in vivo has previously been reported to occur in the rat striatum’. The regional tissue concentration (which approximates intracellular levels) of GABA, taurine and PEA exhibited some differences between the rat strains. The most consistent difference was a significant elevation of aspartate in all regions except the striatum, and deficiencies in tyrosine in all areas. Tissue aspartate is probably localized in both metabolic and transmitter pools, and there is at present no method for estimating these separately. It is thus unknown if the increased content of aspartate derives from one or both pools. However, the generalized increase in this excitant may sensitize the SS rat to epileptogenic stimuli. The extracellular concentration of a neuroactive amino acid is more important than the intracellular level from the neurotransmission point of view. The finding that hippocampal levels of intracellular and stimulated extracellular aspartate are elevated in parallel in the SS strain may be coincidental. No such relationship was seen for taurine and PEA. Furthermore, neither basal nor evoked release of amino acids reflect intracellular concentrations23, and this lack of correlation is also observed during drug-induced seizures”. The SS rat has an abnormally low tissue level of norepinephrine in many brain areas5,14. The underlying biochemical factors responsible for the suppression of norepinephrine have not been identified. They can neither be ascribed to alterations in the activity of the rate-limiting anabolic enzyme tyrosine hydroxylase5, nor to changes in pyridoxal phosphate6, an important regulator of the enzyme. The finding that the norepinephrine precursor ty-
rosine is lower in all regions examined may provide an explanation for the noradrenergic deficiency. While this suggestion warrants further investigation, it should be noted that cerebellar tyrosine is decreased, but norepinephrine is normal’ or eievated14. Huxtable and Laird’” and Huxtable et al.” reported that amino acid concentrations are indistinguishable in SS and SR brains. In contrast, Chapman et al.4 and Ribak et al.” found multiple differences between many amino acids in several areas. However, the results of the latter authors were not totally in agreement, and the present findings show little overlap with previous observations (summarized in Table II). It is indeed unexpected that the results of different laboratories would differ so markedly considering the rather straightforward methodology employed. One possibility is that although the animals originally derive from the same colony, genetic drift may have occurred. Methodological differences are, however, more probably responsible for the discrepancies. The urethane anesthesia used in the present experiments may have affected amino acid levels, and the higher levels of aspartate in SS rats may be secondary to mild hypoglycemia. However, this caveat may not be important, since the amino acid levels reported here are within the normal range. Furthermore, it is unlikely that cerebral amino acids of SS and SR rats would be differently affected by urethane. Age and/or sex differences between animals used in this and previous studies may be of some significance. In conclusion, the supersensitivity of aspartate release after prolonged potassium stimulation, combined with depressed taurine release, may constitute a basis for the low seizure threshold in the SS rat. Furthermore, reduced tissue levels of tyrosine may be partly responsible for the previously reported deficiency in norepinephrine. While it is now evident that the SS rat displays multiple abnormalities in regional amino acid levels, no uniform picture can be obtained when previous and present data are compared. ACKNOWLEDGEMENTS I wish to express my sincere gratitude
to Dr.
137
Leena Tuomisto for making the SS rats available, and to Ms. Birgitta Karlsson for technical assistance. This study was supported by the Ahlen, Ericsson, Wiberg, Swedish Society for Medical Research and Hierta-Retzius Foundations. The support of Dr. Anders Hamberger is gratefully acknowledged (MRC Grant No. 12X-00164).
REFERENCES 1 Andersen, V.E., Genetics in the epilepsies, Trends Neurosci., 8 (1985) 513-516. 2 Bonhaus, D.W. and Huxtable, R.J., Seizure-susceptibility and decreased taurine transport in the genetically epileptic rat, Neurochem. Int., 6 (1984) 365-368. 3 Bonhaus, D. W., Lippincott, SE. and Huxtable, R. J., Subcellular distribution of neuroactive amino acids in brains of genetically epileptic rats, Epilepsia, 25 (1984) 564-568. 4 Chapman, A., Faingold, C.L., Hart, G.P., Bowker, H.M. and Meldrum, B.S., Brain regional amino acid levels in seizure susceptible rats: changes related to sound-induced seizures, Neurochem. Int., 8 (1986) 273-279. 5 Dailey, J.W. and Jobe, P.C., Indices of noradrenergic function in the central nervous system of seizure-naive genetically epilepsy-prone rats, Epilepsia, 27 (1986) 665-670. 6 Ebadi, M., Jobe, P.C. and Laird, II, H.E., The status of vitamin B, metabolism in brains of genetically epilepsy-prone rats, Epilepsia, 26 (1985) 353-359. 7 Faingold, C.L., Meldrum, B.S. and Millan, M.H., Blockade of audiogenic seizure susceptibility by focal injection into the inferior colliculus of an excitant amino acid antagonist, Rr. J. Pharmacol., 84 (1984) 95P. 8 Faingold, CL., Gehlbach, G. and Caspary, D.M., Decreased effectiveness of GABA-mediated inhibition in the inferior colliculus of the genetically epilepsy-prone rat, Exp. Neural., 93 (1986) 145-159. 9 Girault, J.A., Barbeito, L., Spampinato, U., Gozlan, H., Glowinsky, J. and Besson, M.-J., In vivo release of endogenous amino acids from the rat striatum: further evidence for a role of glutamate and aspartate in corticostriatal neurotransmission, J. Neurochem., 47 (1986) 98- 106. 10 Huxtable, R.J. and Laird, II, H.E., Are amino acid patterns necessarily abnormal in epileptic brains? Studies on the genetically seizure-susceptible rat, Neurosci. Lett., 10 (1978) 341-345. 11 Huxtable, R.J. and Laird, H.E., The prolonged anticonvulsant action of taurine on genetically determined seizuresusceptibility, Can. J. Neural. Sci., 5 (1978) 215-221. 12 Huxtable, R.J., Laird, H.E., Bonhaus, D. and Thies, A.C., Correlations between amino acid concentrations in brains of seizure susceptible and seizure resistant rats, Neurochem. Int., 4 (1982) 73-78.
NOTE ADDED IN PROOF I have recently found that extracellular levels of taurine and PEA are particularly sensitive to changes in osmolality. It is therefore possible that the lower Kf-stimulated release of these amino acids in the SS rat hippocampus reflects derangements in osmoregulation.
13 Jobe, P.C., Picchioni, A.L. and Chin, L., Role of brain norepinephrine in audiogenic seizure in the rat, J. Pharmacol. Exp. Ther., 184 (1973) l-10. 14 Jobe, P.C., Laird, II, H.E., Ko, K.H., Ray, T. and Dailey, J.W., Abnormalities in monoamine levels in the central nervous system of the genetically epilepsy-prone rat, Epilepsia, 23 (1982) 359-366. 15 Lehmann, A. and Hamberger,
A., A possible interrelationship between extracellular taurine and phosphoethanolamine in the hippocampus, J. Neurochem., 42 (1984)
1286-1290. 16 Lehmann, A., Sandberg,
M. and Huxtable, R.J., In vivo release of neuroactive amines and amino acids from the hippocampus of seizure-resistant and seizure-susceptible rats, Neurochem.
17 Lehmann,
Int., 8 (1986) 513-520.
A., Alterations in hippocampal extracellular amino acids and purine catabolites during limbic seizures induced by folate injections into the rabbit amygdala, Neuroscience, 22 (1987) 573-578. 18 Lindroth, P., Sandberg, M. and Hamberger, A., Liquid chromatographic determination of amino acids after precolumn fluorescence derivatization. In: A.A. Boulton, G.B. Baker and J.D. Wood (Eds.), Neuromethods: Amino Acids, Vol. 3, Humana Press, Clifton, NJ, 1985, pp. 97-116. 19 Ribak, C.E., Byun, M.Y., Ruiz, G.T. and Reiffenstein, R.J., Increased levels of amino acid neurotransmitters in the inferior colliculus of the genetically epilepsy-prone rat, Epilepsy Res., 2 (1988) 9-13. 20 Roberts, R.C. and Ribak, C.E., Anatomical changes of the GABAergic system in the inferior colliculus of the genetically epilepsy-prone rat, Life Sci., 39 (1986) 789-798. 21 Sandberg, M., Butcher, S.P. and Hagberg, H., Extracellular overflow of neuroactive amino acids during severe insulin-induced hypoglycemia - in vivo dialysis of the rat hippocampus,J. Neurochem., 47 (1986) 178-184. 22 Tacke, U., Audiogenic Seizures in Rats - an Experimental Study of a Genetic Model of Epilepsy, Ph.D. Thesis, University of Kuopio, 1984. 23 Tossman, U., Jonsson, G. and Ungerstedt, U., Regional distribution and extracellular levels of amino acids in rat central nervous system, Acta Physiol. Stand., 127 (1986) 533-545.