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Roles of neurotransmitter amino acids in seizure severity and experience in the genetically epilepsy-prone rat
Brain Research, 560 (1991) 63-70 t~) 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 000689939116970B
63
BRES 16970
Roles of neurotransmitter amino acids in seizure severity and experience in the genetically epilepsy-prone rat Stephen M. Lasley Department of Basic Sciences, University of Illinois College of Medicine at Peoria, Peoria, IL 61656 (U.S.A.) (Accepted 30 April 1991) Key words: Aspartate; Glutamate; ~,-Aminobutyfic acid; Taurine; Genetically epilepsy-prone rat; Seizure experience; Seizure severity; Sound-induced seizure
This investigation was designed to compare seizure-naive and seizure-experienced genetically epilepsy-prone rats (GEPRs) in order to distinguish transmitter amino acid changes related to seizure severity from those associated with seizure experience. Moderate (GEPR-3) and severe (GEPR-9) seizure male GEPRs were divided into seizure-naive and seizure-experienced groups based on whether seizure-inducing acoustical stimuli had been presented between 45 and 60 days of age, and then were sacrificed at 76 -+ 3 days. ~,-Aminobutyric acid (GABA) concentrations were lower in both GEPR-3s and GEPR-9s compared to non-epileptic controls in each brain region examined. Aspartate content was elevated in 5 of 6 brain areas in GEPR-9s compared to non-epileptic controls, and in 3 regions was higher in GEPR-9s than in GEPR-3s. In contrast, tanrine concentrations were higher in GEPR-3s than in non-epileptic controls in each region, and in 4 areas were higher in GEPR-3s than in GEPR-9s. Changes resulting from seizure experience consisted of increases in aspartate, glutamate and giycine in seizure-experienced compared to seizure-naive groups in inferior collicuhis and in motor-sensory and frontal cortices. These findings suggest that the high levels of taurine in GEPR-3s and the elevated content of aspartate in GEPR-9s have roles as determinants of seizure severity. The low concentrations of GABA in both types of GEPRs are consistent with a role for this amino acid in determination of seizure susceptibility. Furthermore, the seizure-induced changes in aspartate and glutamate in both types of GEPRs support the concept that these excitatory amino acids mediate changes in seizure predisposition. The current results are in agreement with previous studies indicating that imbalances in neurotransmitter amino acids are important factors in determining seizure behavior in the GEPR. INTRODUCTION As many as 20% of patients with epilepsy cannot be treated successfully with current antiepileptic agents 2°. Thus, the need remains acute for a better understanding of the multffactorial processes underlying epileptogenesis so that more effective treatments can be devised. Results from numerous animal studies have indicated that excessive excitatory amino acid neurotransmission and/or deficient inhibitory amino acid neuronal activity could play a significant role in the inherited or acquired defects responsible for this condition 5'13'21. The genetically epilepsy-prone rat ( G E P R ) is a useful model for studying the involvement of neurotransmitter amino acids in epileptic mechanisms since an imbalance of excitatory and inhibitory amino acid influences appears to be an underlying factor in seizure initiation and/or predisposition in these animals 12. Accordingly, G E P R s have been utilized in neurochemical, electrophysiological and pharmacological studies examining the roles of these transmitters in seizure behavior. Faingnld et al.13 observed a decreased effectiveness of G A B A
iontophoretically appfied into inferior colliculus of GEPR-9s, leading to the suggestion that G A B A e r g i c inhibitory influences may be deficient at this brain site. Furthermore, Millan et al.23 found that microinjection of G A B A antagonists or excitatory amino acid agonists into inferior colliculus conferred audiogenic seizure susceptibility upon normal rats. Conversely, microinjection of G A B A agonists or excitatory amino acid antagonists into the brainstem auditory pathway up to the inferior colliculus blocked audiogenic seizure susceptibility in G E P R - g s 12'14. This latter work also demonstrated the inferior colliculus to be the most sensitive site for pharmacological manipulation. In agreement with this study, Meldrum et al.22 found that microinjection of an excitatory amino acid antagonist into inferior colliculus, substantia nigra or midbrain reticular formation blocked seizures in GEPR-9s with inferior collicular administration being most effective. Lehmann 19 has reported increases in aspartic acid concentrations in 3 of 4 brain regions from severe seizure G E P R s (GEPR-gs) compared to Sprague-Dawley controls, as well as higher potassium-evoked hippocam-
Correspondence: S.M. Lasley, Department of Basic Sciences, University of Illinois College of Medicine at Peoria, Box 1649, Peoria, IL 61656, U.S.A.
64
Audiogenic Seizure Stimulus
pal a s p a r t a t e release in s e i z u r e - p r o n e rats. In contrast, G A B A and t a u r i n e c o n c e n t r a t i o n s w e r e l o w e r in 2 b r a i n
Seizure-Experienced
p----//---
.......
#1 L. . . . . .
#2 :
#3 L ...........
regions of G E P R - 9 s c o m p a r e d to S p r a g u e - D a w l e y controls. O n the o t h e r h a n d , R i b a k et al. 3° o b s e r v e d large increases in G A B A , in the
central
Age, days
I~ Birth
45
52-53
60
76 Sacrifice
t a u r i n e a n d g l u t a m i c acid c o n t e n t
nucleus
o f the
inferior
colliculus o f
G E P R - 9 s r e l a t i v e to S p r a g u e - D a w l e y controls, in addition to increases in cortical t a u r i n e and g l u t a m a t e in the s e i z u r e - p r o n e rats. T h e s e findings c o r r e s p o n d e d to previous i m m u n o c y t o c h e m i c a l d a t a f r o m t h e s e w o r k e r s ' labo r a t o r y indicating i n c r e a s e d n u m b e r s o f G A B A n e u r o n s
Seizure-Naive
~--//
No Seizure Stimuli Fig. 1. Treatment protocol determining seizure-naive and seizureexperienced genetically epilepsy-prone rats. Seizure-inducing sound stimuli were administered once at 3 weekly intervals between the ages of 45 and 60 days of age to constitute seizure-experienced groups. Seizure-naive groups received no sound stimulation, and were classified on the basis of seizure behavior in littermates.
in i n f e r i o r colliculus of G E P R - 9 s c o m p a r e d to S p r a g u e D a w l e y c o n t r o l s 32. H o w e v e r , C h a p m a n et al. 6 r e p o r t e d l o w e r r e g i o n a l c o n t e n t o f a s p a r t a t e and g l u t a m a t e in a few of the 8 b r a i n areas s a m p l e d f r o m G E P R - 9 s r e l a t i v e to S p r a g u e - D a w l e y
controls, b u t f o u n d that aspartic
acid c o n t e n t i n c r e a s e d in i n f e r i o r colliculus of G E P R - 9 s e x p e r i e n c i n g seizures c o m p a r e d to the s a m e t y p e of animals u n d e r resting conditions. T h e G E P R has an i n h e r i t e d susceptibility to seizures i n d u c e d by e x p e r i m e n t e r - c o n t r o l l e d e v e n t s such as sensory stimuli. Originally d e r i v e d f r o m t h e S p r a g u e - D a w ley strain, the G E P R c u r r e n t l y exists as 2 i n d e p e n d e n t l y d e r i v e d colonies, e a c h exhibiting a characteristic convulsive p a t t e r n in r e s p o n s e to an a u d i t o r y stimulus. M e m b e r s o f the s e v e r e seizure c o l o n y ( G E P R - 9 s )
display
s o u n d - i n d u c e d tonic e x t e n s o r c o n v u l s i o n s , while m e m bers o f the m o d e r a t e seizure c o l o n y ( G E P R - 3 s ) display only clonic c o n v u l s i o n s in r e s p o n s e to the acoustical stimulus. S e i z u r e b e h a v i o r in the. G E P R is also r e s p o n -
degree of seizure predisposition. Members of the moderate seizure GEPR-3 colony exhibit a wild running phase which terminates in generalized clonus following a standardized sound stimulus. Members of the severe seizure GEPR-9 colony exhibit a brief wild running phase that terminates in a tonic extensor convulsion. Non-epileptic controls were the offspring of established seizure-resistant control breeders; these animals never exhibited sound-induced seizures. A screening protocol verifies that each animal exhibits an Audiogenic Response Score characteristic of the colony from which it was derived TM. For this study GEPRs were divided into seizure-naive and seizure-experienced groups based on whether a seizure-inducing acoustical stimulus had been presented. Seizure-experienced GEPRs were sound-stimulated once to induce seizures at each of 3 weekly intervals between 45 and 60 days of age, while seizure-naive GEPRs were classified on the basis of seizures observed in littermates (Fig. 1). Experience indicates that use of this latter criterion ensures a very high probability of assignment of the appropriate seizure phenotype. Non-epileptic controls were divided similarly to GEPRs according to the occurrence or absence of exposure to the acoustical stimulus. Lights in the animal rooms were set on a 12:12 cycle commencing at 06.00 h with temperature maintained at 24 + 1 °C.
sive to a n u m b e r o f a n t i e p i l e p t i c drugs useful in tonicclonic and focal types of e p i l e p s y in m a n 27. T h e f o l l o w i n g study was p e r f o r m e d to c o m p a r e seiz u r e - n a i v e and s e i z u r e - e x p e r i e n c e d G E P R s in an a t t e m p t to distinguish e x c i t a t o r y and inhibitory a m i n o acid transmitter changes
underlying
seizure susceptibility f r o m
t h o s e r e l a t e d to seizure e x p e r i e n c e . M o r e o v e r , G E P R - 3 s w e r e i n c l u d e d in the e x p e r i m e n t a l design to d e t e r m i n e w h e t h e r similar a l t e r a t i o n s in t r a n s m i t t e r a m i n o acids w e r e p r e s e n t in b o t h m o d e r a t e and s e v e r e seizure groups. Finally, p r e v i o u s r e p o r t s o f r e g i o n a l b r a i n a m i n o acid c o n c e n t r a t i o n s in G E P R - 9 s h a v e b e e n s o m e w h a t v a r i a n t , particularly with respect to e x c i t a t o r y a m i n o acids and
GABA.
Tissue dissection Age of the animals at sacrifice was 76 _+ 3 days (mean -+ S.D., range: 69-85 days) with generally one animal drawn per litter (n = 6-9 for each seizure-experienced or seizure-naive group). Rats were sacrificed between 10.00 and 13.00 h by decapitation with immediate immersion in liquid nitrogen for 5-6 s. Brains were quickly removed and immediately dissected in a cryostat maintained at -5 °C using the atlas of Paxinos and Watson 26 as a guide. Frontal cortex was removed from the medial half of each hemisphere rostral to the genu of the corpus callosum. Striatum was dissected from a coronal slice bounded by cuts through the head of the eaudate and through the rostral edge of the thalamus. Motorsensory cortex was defined as that cortex overlying the striatum and hippocampus minus dorsomedial (cingulate and occipital) and ventrolateral (pyriform and entorhinal) portions. Thalamus was dissected from a coronal slice bounded by cuts through the rostral edges of this structure and that of the medial geniculate. Hippocampus and inferior collieulus were removed on the basis of observable landmarks. Tissue was stored at -75 °C until analysis.
MATERIALS AND METHODS
Animals Four types of rats were employed in this study. Commercially bred Sprague-Dawley males were obtained from Harlan SpragueDawley, Inc. (Indianapolis, IN) at 65 days of age and utilized as one type of control subject. The other rats were males obtained from 3 independent colonies maintained at the University of Illinois College of Medicine at Peoria. Each colony has been separately derived from Sprague-Dawley stock for a specific type and
Sample preparation and chromatography Amino acids were quantified essentially according to Cohen et al. 8 and Bidlingmeyer et al. 3. Brain regions were homogenized in 20-30 vols of 0.1 M HC1 (constant volume for a given region) and ultrafiltered by centrifugation. Methionine sulfone in 0.1 M HCI was added as an internal standard to an aliquot of each extract, and this mixture was dried under vacuum prior to derivatization with phenylisothioeyanate and a final drying step. Derivatives were redissolved in the initial mobile phase, placed in an automated
65 sample injector and analyzed by binary gradient liquid chromatography with detection by ultraviolet absorbanee at 254 nm (PICOTAG Amino Acid System, Waters Chromatography Div., Milford, MA). Flow rate was 1.0 ml/min. The amino acids of interest - aspartie and glutamie acids, G A B A , taurine and glycine - were quantitated by integration of peak areas with comparison to standards of known concentration included in each run. Tissue pellets from the initial centrifugation were dissolved in 1.0 M NaOH and protein concentrations determined by the method of Smith et al.33 using bovine serum albumin as a standard. Bovine serum albumin, methionine sulfone and amino acid standards were obtained from Sigma Chemical Co. (St. Louis, MO). Mobile phases for the gradient chromatography were obtained directly from Waters Chromatograpy Div. (Milford, MA).
tiates the action of glutamate at an allosteric site on one type of excitatory amino acid receptor, and because its role as an inhibitory transmitter in mammalian forebrain is r e l a t i v e l y m i n o r . T h e m o s t c o n s i s t e n t o b s e r v a t i o n w a s t h a t in 5 o f 6 b r a i n a r e a s a s p a r t i c acid c o n c e n t r a t i o n s w e r e significantly h i g h e r i n G E P R - 9 s c o m p a r e d t o n o n e p i l e p t i c c o n t r o l s in t h e r a n g e o f 7 - 1 2 % , e x c e p t f o r f r o n tal c o r t e x in w h i c h t h e e l e v a t i o n w a s 41%. I n c o n t r a s t , in 2 r e g i o n s in G E P R - 3 s - h i p p o c a m p u s a n d t h a l a m u s a s p a r t i c acid c o n t e n t w a s significantly less t h a n i n n o n epileptic controls. Abnormalities
Data analysis Amino acid concentrations were analyzed by two-factor analyses of variance for each amino acid in each brain region with seizure severity (non-epileptic control, GEPR-3, GEPR-9) and seizure experience as the main factors. Standard t-tests were utilized to determine the bases of significant effects by examining a set of group comparisons established a priori.
in g l u t a m i c acid c o n c e n t r a t i o n s w e r e
less n o t a b l e in G E P R - 9 s c o m p a r e d t o n o n - e p i l e p t i c c o n t r o l s w i t h significant i n c r e a s e s o n l y in h i p p o c a m p u s a n d f r o n t a l c o r t e x . O n t h e o t h e r h a n d , g l u t a m i c acid c o n t e n t in G E P R - 3 s
w a s significantly h i g h e r in f r o n t a l c o r t e x ,
b u t w a s l o w e r in s t r i a t u m c o m p a r e d t o n o n - e p i l e p t i c c o n trols. F e w e r s i g n i f i c a n t c h a n g e s in glycine c o n c e n t r a t i o n s
RESULTS
w e r e p r e s e n t : in G E P R - 9 s g l y c i n e w a s e l e v a t e d in h i p p o c a m p u s a n d d i m i n i s h e d in f r o n t a l c o r t e x r e l a t i v e to c o n -
Aspartate, glutamate, glycine
trois, w h i l e in G E P R - 3 s
glycine w a s l o w e r in s t r i a t u m .
T a b l e I gives t h e r e g i o n a l b r a i n a m i n o acid c o n c e n t r a -
In 3 brain regions (hippocampus, striatum and thala-
t i o n s f o r a s p a r t a t e , g l u t a m a t e a n d glycine a c c o r d i n g t o
m u s ) a s p a r t a t e a n d g l u t a m a t e c o n c e n t r a t i o n s w e r e sig-
s e i z u r e p h e n o t y p e . G l y c i n e is i n c l u d e d b e c a u s e it p o t e n -
n i f i c a n t l y s m a l l e r in G E P R - 3 s t h a n in G E P R - 9 s . A n a l o -
TABLE I
Regional brain excitatory amino acid concentrations by seizure severity Values are expressed as mean ± S.D. in nmol/mg protein with n = 11-17 for each mean. Concentrations from seizure-experienced and seizure-naive animals are combined to show the bases of statistical significance with respect to seizure severity.
Region
Animal
ASP
GL U
GLY 1
Hippocampus
N-E control GEPR-3 GEPR-9
30.5 ± 3.3 27.6 ± 2.8 *'c 34.3 ± 4.7*
113.1 ± 11.8 104.9 ± 10.5 b 124.3 ± 15.1"
11.1 ± 1.4 10.3 ± 0.9 b 12.1 ± 1.5"
Inferior colliculus
N-E control GEPR-3 GEPR-9
32.5 ± 2.7 34.8 -+ 4.7 35.9 ± 3.6**
Motor-sensory cortex
N-E control GEPR-3 GEPR-9
Frontal cortex
68.2 ± 5.4 72.3 ± 7.7 73.1 ± 8.3
18.8 ± 1.0 19.3 ± 1.7 19.9 ± 1.8
46.4 ± 4.0 46.1 ± 4.0 44.1 ± 4.2
114.8 ± 11.0 119.2 ± 11.6 112.9 ± 8.7
8.1 ± 0.8 8.6 ± 0.7 8.2 ± 0.8
N-E control GEPR-3 GEPR-9
39.0 ± 7.1 47.7 ± 12.6 54.9 ± 8.5***
270.3 ± 29.5 314.3 -- 33.0** 296.2 ± 32.1'
Anterior striatum
N-E control GEPR-3 GEPR-9
23.9 ± 2.9 23.9 - 2.3 a 26.6 ± 2.8*
94.3 ± 8.4 84.9 -+ 7.7 **'b 97.5 ± 12.0
8.7 ± 0.7 7.9 ± 1.1 *'a 8.8 ± 0.9
Thalamus
N-E control GEPR-3 GEPR-9
29.2 ± 2.4 24.4 ± 2.9 ***'¢ 31.3 ± 2.9*
73.6 --- 10.6 67.6 ± 6.9 b 77.2 + 8.1
9.5 ± 0.9 9.5 ± 0.9 9.7 - 0.6
ASP, Aspartic acid; GLU, glutamic acid; GLY, glycine. t Giycine is included because it is active at an allosteric site on one subtype of glutamate receptor. ap < 0.05; bp < 0.01; cp < 0.001 compared to GEPR-9 value *P < 0.05; **P < 0.01; ***P < 0.001 compared to the non-epileptic (N-E) control value.
26.3 ± 2.5 26.6 ± 2.1 b 24.4 ± 1.4"
66 gous changes in glycine were noted in 2 areas (Table I). Table II displays the statistically significant changes in aspartate, glutamate and glycine concentrations that occurred as a result of seizures induced by sound stimulation at 3 weekly intervals in G E P R - 3 s and G E P R - 9 s . Increases in content of these amino acids were observed in inferior colliculus of seizure-experienced G E P R - 9 s in comparison to seizure-naive rats of the same seizure phenotype. In addition, elevations in aspartic acid in seizure-experienced G E P R - 9 s were also found in frontal cortex. Changes in these amino acids were noted in seizure-experienced c o m p a r e d to seizure-naive G E P R - 3 s in both cortical areas sampled: glutamate and glycine were increased in motor-sensory cortex as a result of seizure experience while glutamate and aspartate were increased in frontal cortex. With the exception of the change in aspartic acid in frontal cortex (35%), the other increases induced by seizure experience were in the range of 1020%.
In contrast, taurine concentrations were significantly higher in G E P R - 3 s c o m p a r e d to non-epileptic controls in the range of 14-57% in all brain areas sampled. However, taurine content in G E P R - 9 s was significantly higher relative to non-epileptic controls only in hippocampus. Thus, taurine levels in the 2 types of G E P R could be distinguished statistically in 4 brain regions in which values in G E P R - 3 s were higher - inferior colliculus, motor-sensory and frontal cortices and thalamus. Only 2 statistically significant changes in G A B A and taurine concentrations resulted from seizures induced by sound stimulation in G E P R - 3 s and G E P R - 9 s at the 3 weekly intervals. G A B A content in thalamus was higher in seizure-experienced than in seizure-naive G E P R - 9 s , while taurine concentrations in frontal cortex were higher in seizure-experienced than in seizure-naive G E P R - 3 s (data not shown). N o n - e p i l e p t i c c o n t r o l s vs S p r a g u e - D a w l e y
controls
The decreases in G A B A and increases in aspartic acid observed throughout the brains of G E P R - 9 s (Tables I and III) p r e s e n t e d a p p a r e n t discrepancies with previous reports by other laboratories 6"3°'32. Since the experimen-
Taurine, G A B A
Table III gives the regional brain amino acid concentrations for taurine and G A B A according to seizure phenotype. Consistent changes were o b s e r v e d in b o t h these amino acids. G A B A content was significantly lower in both G E P R - 3 s and G E P R - 9 s c o m p a r e d to non-epileptic controls in the range of 12-33% in all brain regions examined. H o w e v e r , there was little differentiation between the 2 types of seizure-prone rats in the magnitude of the decreases in G A B A . G E P R - 3 s and G E P R - 9 s were distinguishable statistically in only 2 brain areas: G E P R - 3 s had significantly lower concentrations of G A B A than G E P R - 9 s in hippocampus, but this relationship was reversed in frontal cortex.
tal subjects (i.e. G E P R - 9 s ) were p r e s u m a b l y identical in each investigation, the selection of control animals for comparison to G E P R s was considered to be one potential differentiating factor. Consequently, 8 commercially b r e d male S p r a g u e - D a w l e y rats were purchased for comparison of regional amino acid concentrations to those in a separate group of 7 non-epileptic controls. The non-epileptic control colony has been b r e d alongside the G E P R colonies to be free of seizure behavior and has been the source of all other control animals utilized in this study.
TABLE II Regional changes in excitatory amino acids resulting from seizure experience
Values are expressed as mean - S.D. in nmol/mg protein ith n = 6-9 for each mean. Region
ASP
GL U
GL Y 1
SN ~ SE
SN ~ SE
SN ~ SE
GEPR-3 GEPR-9
33.7 +- 1.8 --* 38.0 --- 3.6*
-
Motor-sensory cortex
GEPR-3 GEPR-9
-
111.0 -- 7.4 ~ 125.3 --- 10.6" -
8.1 _+ 0.5 ~ -
Frontal cortex
GEPR-3 GEPR-9
40.2 _+ 8.7 ~ 54.4 +- 12.0" 49.4 - 6.7 ~ 59.7 - 7.1"
293.8 - 27.0 --~ 332.2 +- 27.6* -
-
Inferior coiliculus
Animal
68.6 +- 3.5 ~
77.0 --- 9.5*
18.9 -+ 0.9 ~ 20.7 _+ 1.9" 8.9 -+ 0.6*
ASP, Aspartic acid; GLU, glutamic acid; GLY, glycine; SN, mean for seizure-naive animals; SE, mean for seizure-experienced animals; -, no change. i Glycine is included because it is active at an allosteric site on one subtype of glutamate receptor. *P < 0.05 compared to seizure-naive value.
67 TABLE III
d e t e r m i n a t i o n s are s h o w n in T a b l e IV. M o s t n o t a b l e was
Regional brain inhibitory amino acid concentrations by seizure severity
t h e finding that in e a c h b r a i n a r e a s a m p l e d G A B A cont e n t in S p r a g u e - D a w l e y c o n t r o l s was significantly less t h a n that in n o n - e p i l e p t i c controls. T a u r i n e c o n c e n t r a -
Values are expressed as mean -+ S.D. in nmol/mg protein with n = 11-17 for each mean. Concentrations from seizure-experienced and seizure-naive animals are combined to s h o w the bases of statistical significance with respect to seizure severity.
tions w e r e significantly less in s t r i a t u m and i n f e r i o r colliculus o f S p r a g u e - D a w l e y
c o m p a r e d to n o n - e p i l e p t i c
c o n t r o l s , as w e r e a s p a r t a t e , g l u t a m a t e and glycine con-
Region
Animal
TA U
GABA
c e n t r a t i o n s in i n f e r i o r colliculus.
Hippocampus
N-E control GEPR-3 GEPR-9
86.0 -+ 10.4 106.2 - 9.4*** 99.0 _ 11.0"**
19.2 - 2.4 13.8 -+ 1.9 ***'b 16.8 -+ 2.6*
DISCUSSION
Inferior collieulus
N-E control GEPR-3 GEPR-9
33.8 -+ 2.9 53.2 -+ 5.1 ***'c 33.4 - 2.6
25.4 - 3.0 21.9 -+ 3.0*** 21.2 -+ 3.1"**
studies h a v e d e f i n e d an a p p a r e n t i m b a l a n c e o f e x c i t a t o r y
Motor-sensory N-E control cortex GEPR-3 GEPR-9
82.2 -+ 7.6 112.2 _+ 6.9 ***'c 79.0 - 5.9
12.3 - 2.1 9.7 - 1.8"** 9.7 -+ 1.2"**
e v e r , the results o f n e u r o c h e m i c a l investigations h a v e
Frontal cortex
Anterior striatum
Thalamus
N-E control GEPR-3 GEPR-9 N-E control GEPR-3 GEPR-9 N-E control GEPR-3 GEPR-9
Previous
37.5 -+ 3.0 48.7 _ 4.2 ***'c 39.0 -+ 2.9
and
electrophysiological
and i n h i b i t o r y a m i n o acid n e u r o t r a n s m i s s i o n in G E P R brain, particularly in i n f e r i o r colliculus 12-14'22'23. H o w n o t clearly s u p p o r t e d t h e s e findings 6'3°. T h e c u r r e n t study has p r o v i d e d a d d i t i o n a l clarification o f t h e roles of t h e s e
196.1 -+ 24.6 36.2 -+ 3.8 288.5 - 30.4 ***'c 30.7 - 3.0 ***'b 206.5 -+ 11.6 27.4 -+ 3.1'** 118.5 _+ 11.4 134.5 -+ 15.0"* 125.4 -+ 11.5
pharmacological
a m i n o acids by the inclusion o f s e i z u r e - n a i v e as well as s e i z u r e - e x p e r i e n c e d m o d e r a t e a n d s e v e r e seizure G E P R s a l o n g with a c o m p a r i s o n o f 2 types o f c o n t r o l animals.
22.8 -+ 3.1 15.2 _+ 2.0*** 16.1 -+ 3.l***
T h e results suggest that the high c o n c e n t r a t i o n s o f taurine in G E P R - 3 s and the e l e v a t e d c o n t e n t o f aspartic acid in G E P R - 9 s h a v e roles as d e t e r m i n a n t s o f seizure
13.2 - 2.0 10.1 -+ 1.6"** 10.8 -+ 1.4"**
severity. T h e low c o n c e n t r a t i o n s of G A B A in b o t h types of G E P R s indicate a r o l e for this t r a n s m i t t e r in determination
TAU, Taurine; GABA, y-aminobutyric acid. ap < 0.05; bp < 0.01; cp < 0.001 compared to GEPR-9 value. *P < 0.05; **P < 0.01; ***P < 0.001 compared to the nonepileptic (N-E) control value.
of
seizure
susceptibility.
In
addition,
the
c h a n g e s in a s p a r t a t e a n d g l u t a m a t e levels i n d u c e d by seizures in specific b r a i n r e g i o n s in b o t h types o f G E P R s suggest that t h e s e e x c i t a t o r y a m i n o acids m e d i a t e changes resulting f r o m seizure e x p e r i e n c e . Finally, m o d e r a t e seiz u r e G E P R - 3 s h a v e b e e n s h o w n to b e d i f f e r e n t f r o m se-
A n i m a l s o f t h e s e c o n t r o l g r o u p s w e r e sacrificed at
v e r e seizure G E P R - 9 s in t e r m s o f h i g h e r taurine and
ages similar to t h o s e o f the o t h e r animals e m p l o y e d in
l o w e r aspartic and g l u t a m i c acid c o n c e n t r a t i o n s .
this study, and i n f e r i o r colliculus, a n t e r i o r s t r i a t u m and motor-sensory
c o r t e x dissected and p r o c e s s e d
T h e w i d e s p r e a d r e g i o n a l increases in a s p a r t a t e a n d
as de-
d e c r e a s e s in G A B A
scribed in M a t e r i a l s a n d M e t h o d s . T h e results o f t h e s e
o b s e r v e d in G E P R - 9 s in this w o r k
are in g e n e r a l a g r e e m e n t with the changes in tissue
TABLE IV Comparison of regional amino acid content in non-epileptic and Sprague-Dawley controls Values are expressed as mean - S.D. in nmol/mg protein with n = 7-8 for each mean. Region
Animal
ASP
GL U
GLY
Inferior eolliculus
N-E control SD control
31.3 -+ 2.6 26.5 - 2.7*
70.1 - 5.6 61.8 -+ 4.6**
20.8 -+ 1.4 18.1 - 1.2"*
Anterior striatum
N-E control SD control
22.6 -+ 2.9 21.1 - 3.1
92.0 --_ 8.9 93.3 -+ 11.1
9.0 -+ 0.6 8.9 -+ 1.1
Motor-sensory cortex
N-E control SD control
43.2 + 3.9 46.0 + 4.6
116.7 -+ 12.0 128.5 + 9.3
8.2 -+ 1.0 9.4 + 0.5**
TA U 31.6 --- 3.1 24.2 - 1.8"**
GABA 25.1 _+ 2.8 18.6 -+ 1.5"**
122.7 - 11.9 104.0 _+ 8.0**
23.6 -+ 3.2 18.4 _+ 2.1"*
80.3 - 8.6 82.9 -+ 7.0
12.6 + 2.3 10.4 _+ 1.1"
N-E control, non-epileptic control animals; SD control, Spragne-Dawley control animals. See legends of Tables I and III for amino acid abbreviations. *P < 0.05; **P < 0.01; ***P < 0.001 compared to the non-epileptic control value.
68 amino acid levels reported by Lehmann 19. Tissue amino acid concentrations approximate those levels present intracellularly, but tissue aspartate (and glutamate) is undoubtedly distributed between metabolic and transmitter pools. Currently, there are no means to accurately define the contributions to total aspartate from each source, and thus it cannot be determined whether the increases in aspartic acid emanate from one or both sources. Nonetheless, one would anticipate that a general increase in this excitatory amino acid in conjunction with a general decrease in G A B A would promote the initiation and propagation of seizures. In contrast with the increased aspartate content and decreased G A B A concentrations found in inferior colliculus in GEPR-9s in the current study (Tables I and III), Ribak et al. 3° reported significant increases in G A B A , glutamate and taurine in the central nucleus of this structure in severe seizure rats. This apparent discrepancy can be at least partially resolved by consideration of the types of control animals employed. Ribak et al. 30 utilized commercial Sprague-Dawley rats as controls, and by comparison of the data in Tables I, III and IV it is reasonable to conclude that GEPR-9s have significantly greater levels of aspartate, glutamate, glycine (P < 0.05), taurine and G A B A (P < 0.05) relative to Sprague-Dawley controls. Direct statistical comparisons of GEPR-9s and Sprague-Dawley control values were made bearing in mind that these tissues were assayed at different time periods and the means were based on markedly different sample sizes (i.e., 17 and 8, respectively). Other factors may account for differences between the findings of the current investigation and those of Chapman et al. 6. These workers reported elevations in aspartate content in inferior colliculus of GEPR-9s undergoing seizures compared to GEPR-9s at rest, though interictal levels of aspartate and glutamate were lower in some brain areas of GEPR-9s relative to Sprague-Dawley controls. However, these latter amino acid concentrations could have been reduced by close proximity of the last seizure with sacrifice of the animals for analyses. The inferior colliculus is the most important structure in the auditory pathway of the GEPR with respect to audiogenic seizures. It is the site most sensitive to pharmacological manipulations of excitatory amino acid or GABAergic influences 13'14"22. Moreover, infusion of an excitatory amino acid or a G A B A antagonist into inferior colliculus renders a significant proportion of normal rats susceptible to audiogenic seizures 23. A deficiency of neuronal inhibition has also been reported to be present in this structure ~3. It is reasonable then to suggest that the elevated concentrations of aspartic acid (GEPR-9s) and lower amounts of G A B A (GEPR-3s and GEPR-9s) observed in inferior colliculus in this work predispose this site to
seizure initiation and spread as a result of sound stimulation. Concentrations of taurine were higher in all brain regions in GEPR-3s compared to non-epileptic controls as shown in Table III, but changes were generally absent in GEPR-9s. Alterations in taurine content have been reported by others in GEPR-9s 19 but have not been consistently observed across studies. Taurine has been shown to enhance glutamate metabolism in GEPRs 4 and to possess potent anticonvulsant properties when administered intracerebroventricularly or into inferior colliculus in these animals Lr. However, except for specific sites in the brain - such as retina or hippocampal pyramidal cells where taurine appears to act as a transmitter, it serves primarily a neuromodulatory or osmoregulatory role 17. Nonetheless, its anticonvulsant effects suggest that the increased taurine concentrations in GEPR-3s may be an important factor in compensating for the diminished levels of G A B A and thus play a role in determining seizure severity. The most striking results of this study were the statistically significant increases in aspartate, glutamate and glycine concentrations that occurred in inferior colliculus and motor-sensory and frontal cortices in GEPR-3s and/or GEPR-9s (Table II) as a result of the induction of seizures at 3 weekly intervals 16-31 days prior to sacrifice. However, the processes underlying these increases in amino acid content are not apparent. Kindling stimulation in rats causes hippocampal mossy fibers to establish aberrant synaptic contacts (i.e., causes sprouting) even before generalized seizures o c c u r 34. This process is accompanied by increases in kainate receptor binding 29, and presumably glutamate-containing synapses, in the innervated areas. It is known that in GEPRs a lower proportion of animals exhibit their phenotypic seizure behavior on the first exposure to sound stimulation than on the third exposure 24'25"3L, suggesting the presence of a type of kindling. Reigel et al. 2s have shown that this effect of seizure experience occurs independently of the development of seizure susceptibility or severity. Furthermore, Ben-Aft and Gho 2 have reported that brief epileptiform episodes induced in hippocampal slices generate a persistent potentiation of synaptic transmission with characteristics similar to those of long-term potentiation. Presynaptic changes - such as increased aspartate/glutamate release - constitute a major component of the long-term potentiation phenomenon 1"7. Moreover, the findings of Davies et al. x~ in hippocampus suggest that a deficiency in GABAergic inhibition may predispose to this synaptic potentiation. However, the processes of long-term potentiation and sprouting have not been generalized to brain regions such as inferior colliculus and thus may not completely account for these ob-
69 servations. Nonetheless, the increased concentrations of aspartate, glutamate and glyeine induced by seizure experience - especially in the inferior colliculus - would be expected to further promote seizure behavior induced by sound stimulation. Extensive comparisons of regional brain biogenic amine concentrations in GEPR-3s and GEPR-9s have been made previously 9'1°. Deficits in norepinephrine and 5-hydroxytryptamine content were found in both types of G E P R in most areas sampled and were of graded or similar magnitude. The transmitter amino acid profiles in moderate and severe seizure GEPRs in Tables I-III present a different picture, however. While the changes resulting from seizure experience and the decreases in G A B A content were generally of similar magnitude in both types of G E P R , other alterations were more characteristic of one group or the other. Increases in taurine were generally typical of GEPR-3s - in 4 of the 6 brain areas taurine concentrations were significantly greater in these animals than in GEPR-9s. On the other hand, increases in aspartic acid were generally typical of GEPR9s, and in 3 brain regions aspartic and glutamic acid levels were significantly greater in these animals than in GEPR-3s. These findings suggest that aspartate, glutamate and taurine play important roles in distinguishing moderate and severe seizure GEPRs in combination with other reported distinctions between the 2 groups, such as greater elevations in hearing thresholds in GEPR3s 15. Thus, differing patterns of biochemical and physiological changes may underlie the graded seizure severity of these 2 groups. Table V presents a summary of the relative changes observed in transmitter amino acid cOncentrations in this study and relates these findings to qualitative differences in seizure behavior in the G E P R and 2 types of controls involved. Both GEPR-3s and GEPR-9s originated from commercially-bred Sprague-Dawley rats, and it has been
observed that a small proportion of the latter animals exhibit a running response and/or clonic convulsion in the presence of the sound stimulus that induces seizures in GEPRs 27. The non-epileptic controls rarely exhibit seizure behavior, and animals that do are continually excluded from the colony. Non-epileptic controls had the highest levels of G A B A in this investigation coupled with comparatively lower concentrations of aspartate, suggesting a relative dominance of inhibitory amino acid influences commensurate with their inbred seizure-resistant character. In comparison, Sprague-Dawley controls exhibited lower levels of G A B A but similar concentrations of aspartic and glutamic acids to non-epileptic controis except in inferior colliculus where both aspartate and glutamate values were lower. Thus, Sprague-Dawley controls would be expected to be somewhat more susceptible to seizure-inducing stimuli than non-epileptic controls on the bases of these findings. Both types of G E P R possess lower levels of G A B A than non-epileptic controls and demonstrate increases in aspartate and/or glutamate as a result of seizure experience. These changes suggest a relative dominance of excitatory amino acid influences in GEPRs commensurate with their seizure-prone nature. The high amounts of taurine in GEPR-3s and aspartic acid in GEPR-9s may contribute to the distinction between these 2 groups with respect to moderate and severe seizure intensity, respectively. Thus, the results of this work are in general agreement with pharmacological and electrophysiological studies which have suggested that an imbalance of excitatory and inhibitory amino acid influences at least partially accounts for the seizure-prone nature of GEPRs. In conclusion, this investigation has shown that relative to non-epileptic controls the seizure-prone nature of GEPRs is associated with generally low concentrations of G A B A , while seizure severity is differentiated at least partially by abnormalities in the levels of taurine (GEPR-
TABLE V Correlation o f amino acid changes with seizure behavior
See legends of Tables I, III and IV for listing of abbreviations. IC, Inferior collieulus; MSC, motor-sensory cortex; FC, frontal cortex. N-E controls
Amino acid characteristics
Seizure-behavior
SD controls
GEPR-3
GEPR-9
GABA
Highest
< N-E controls
Low
Low
TAU
-
-
H i g h e s t
-
ASP, GLU
-
< N-E controls (IC)
-
Highest ASP
GLU increases with seizure experience (MSC, FC)
ASP, GLU increase with seizure experience (IC, FC)
Seizureprone, moderate intensity
Seizure prone, severe intensity
Non-epileptic
Very low incidence, minimal intensity
70 3s) o r a s p a r t a t e ( G E P R - 9 s ) . O n the o t h e r h a n d , induc-
the functional nature of these n e u r o c h e m i c a l changes.
tion o f m u l t i p l e seizure e p i s o d e s in G E P R s p r o d u c e s regionally-specific increases in a s p a r t a t e ,
glutamate
and
glycine associated with an e n h a n c e m e n t of seizure predisposition. F u r t h e r e x p e r i m e n t a t i o n is necessary to confirm
Acknowledgements. The author wishes to express his gratitude to Mary Green and Robert Burger for excellent technical assistance in the conduct of this work.
REFERENCES 1 Bekkers, J.M. and Stevens, C.E, Presynaptic mechanism for long-term potentiation in the hippocampus, Nature, 346 (1990) 724-729. 2 Ben-Ari, Y. and Gho, M., Long-lasting modification of the synaptic properties of rat CA3 hippocampal neurones induced by kainic acid, J. Physiol., 404 (1988) 365-384. 3 Bidlingmeyer, B.A., Cohen, S.A. and Tarvin, T.L., Rapid analysis of amino acids using pre-column derivatization, J. Chromatogr., 336 (1984) 93-104. 4 Bonhaus, D.W. and Huxtable, R.J., The transport, biosynthesis, and biochemical actions of taurine in a genetic epilepsy, Neurochem. Int., 5 (1983) 413-419. 5 Chapman, A.G., Anticonvulsant activity of excitatory amino acid antagonists. In E.A. Cavalheiro, J. Lehmann and L. Turski (Eds.)~ Frontiers in Excitatory Amino Acid Research, Alan R. Liss, New York, 1988, pp. 203-210. 6 Chapman, A.G., 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. Intl., 8 (1986) 273-279. 7 Collingridge, G.L. and Davies, S.N., NMDA receptors and long-term potentiation in the hippocampus. In J.C. Watkins and G.L. Collingridge (Eds.), The NMDA Receptor, Oxford Univ. Press, New York, 1989, pp. 123-136. 8 Cohen, S.A., Bidlingmeyer, B.A. and Tarvin, T.L., PITC derivatives in amino acid analysis, Nature, 320 (1986) 769-770. 9 Dailey, J.W., Reigel, C.E., Mishra, P.K. and Jobe, P.C., Neurobiology of seizure predisposition in the genetically epilepsyprone rat, Epilepsy Res., 3 (1989) 3-17. 10 Dailey, J.W., Mishra, P.K., Ko, K.H., Penny, J.E. and Jobe, P.C., Noradrenergic abnormalities in the central nervous system of seizure-naive genetically epilepsy-prone rats, Epilepsia, 32 (1991) 168-173. 11 Davies, C.H., Starkey, S.J., Pozza, M.F. and Collingridge, G.L., GABA B autoreceptors regulate the induction of LTP, Nature, 349 (1991) 609-611. 12 Faingold, C.L., The genetically epilepsy-prone rat, Gen. Pharmacol., 19 (1988) 331-338. 13 Faingold, C.L., Gehlbach, G. and Caspary, D.M., Decreased effectiveness of GABA-mediated inhibition in the inferior colliculus of the genetically epilepsy-prone rat, Exp. Neurol., 93 (1986) 145-159. 14 Faingold, C.L., Millan, M.H., Boersma, C.A. and Meldrum, B.S., Excitant amino acids and audiogenic seizures in the genetically epilepsy-prone rat, Exp. Neurol., 99 (1988) 678-686. 15 Faingold, C.L., Walsh, E.J., Maxwell, J.K. and Randall, M.E., Audiogenic seizure severity and hearing deficits in the genetically epilepsy-prone rat, Exp. Neurol., 108 (1990) 55-60. 16 Huxtable, R.J., Insights on function: metabolism and pharmacology of taurine in the brain. In J.B. Lombardini and A. Kenny (Eds.), The Role of Peptides and Amino Acids as Neurotransmitters), Alan R. Liss, New York, 1981, pp. 53-97. 17 Huxtable, R.J., Taurine in the central nervous system and the mammalian actions of taurine, Prog. Neurobiol., 32 (1989) 471533. 18 Jobe, P.C., Picchioni, A.L. and Chin, L., Role of brain nore-
19
20
21
22
23
24
25
26 27
28
29
30
31
32
33
34
pinephrine on audiogenic seizure in the rat, J. Pharmacol. Exp. Ther., 184 (1973) 1-10. Lehmann, A., Abnormalities in the levels of extracellular and tissue amino acids in the brain of the seizure-susceptible rat, Epilepsy Res., 3 (1989) 130-137. Lrscher, W. and Schmidt, D., Which animal models should be used in the search for new antiepileptic drugs? A proposal based on experimental and clinical considerations, Epilepsy Res., 2 (1988) 145-181. Meldrum, B., Excitatory amino acids and epilepsy. In T.P. Hicks, D. Lodge and H. McLennan (Eds.), Excitatory Amino Acid Transmission, Alan R. Liss, New York, 1987, pp. 189-196. Meldrum, B., Millan, M., Patel, S. and de Sarro, G., Anti-epileptic effects of focal microinjection of excitatory amino acid antagonists, J. Neural Transm., 72 (1988) 191-200. Millan, M.H., Meldrum, B.S. and Faingold, C.L., Induction of audiogenic seizure susceptibility by focal infusion of excitant amino acid or bicuculline into the inferior colliculus of normal rats, Exp. Neurol., 91 (1986) 634-639. Mishra, P.K., Dailey, J.W., Reigel, C.E., Tomsic, M.L. and Jobe, P.C., Sex-specific distinctions in audiogenic convulsions exhibited by severe seizure genetically epilepsy-prone rats (GEPR-9s), Epilepsy Res., 2 (1988) 309-316. Mishra, P.K., Dailey, J.W., Reigel, C.E. and Jobe, P.C., Audiogenic convulsions in moderate seizure genetically epilepsyprone rats (GEPR-3s), Epilepsy Res., 3 (1989) 191-198. Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, Orlando, FL, 1986. Reigel, C.E., Dailey, J.W. and Jobe, P.C., The genetically epilepsy-prone rat: an overview of seizure-prone characteristics and responsiveness to anticonvulsant drugs, Life Sci., 39 (1986) 763-774. Reigel, C.E., Jobe, P.C., Dailey, J.W. and Savage, D.D., Ontogeny of sound-induced seizures in the genetically epilepsyprone rat, Epilepsy Res., 4 (1989) 63-71. Represa, A., Le Gal La Salle, G. and Ben-Ari, Y., Hippocampal plasticity in the kindling model of epilepsy in rats, Neurosci. Lett., 99 (1989) 345-350. Ribak, C.E., Byun, M.Y., Thomas Ruiz, G. 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. Ribak, C.E., Roberts, R.C., Byun, M.Y. and Kim, H.L., Anatomical and behavioral analyses of the inheritance of audiogenic seizures in the progeny of genetically epilepsy-prone and Sprague-Dawley rats, Epilepsy Res., 2 (1988) 345-355. Roberts, R.C., Ribak, C.E. and Oertel, W.H., Increased numbers of GABAergic neurons in the inferior coUiculus of an audiogenic model of genetic epilepsy, Brain Research, 361 (1985) 324-338. Smith, P.K., Krohn, R.I., Hermanson, G.T,, Mallia, A.K., Gartner, EH., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J. and Klenk, D.C., Measurement of protein using bicinchoninic acid, Anal. Biochem., 150 (1985) 76-85. Sutula, T., Xiao-Xian, H., Cavazos, J. and Scott, G., Synaptic reorganization in the hippocampus induced by abnormal functional activity, Science, 239 (1988) 1147-1150.
63
BRES 16970
Roles of neurotransmitter amino acids in seizure severity and experience in the genetically epilepsy-prone rat Stephen M. Lasley Department of Basic Sciences, University of Illinois College of Medicine at Peoria, Peoria, IL 61656 (U.S.A.) (Accepted 30 April 1991) Key words: Aspartate; Glutamate; ~,-Aminobutyfic acid; Taurine; Genetically epilepsy-prone rat; Seizure experience; Seizure severity; Sound-induced seizure
This investigation was designed to compare seizure-naive and seizure-experienced genetically epilepsy-prone rats (GEPRs) in order to distinguish transmitter amino acid changes related to seizure severity from those associated with seizure experience. Moderate (GEPR-3) and severe (GEPR-9) seizure male GEPRs were divided into seizure-naive and seizure-experienced groups based on whether seizure-inducing acoustical stimuli had been presented between 45 and 60 days of age, and then were sacrificed at 76 -+ 3 days. ~,-Aminobutyric acid (GABA) concentrations were lower in both GEPR-3s and GEPR-9s compared to non-epileptic controls in each brain region examined. Aspartate content was elevated in 5 of 6 brain areas in GEPR-9s compared to non-epileptic controls, and in 3 regions was higher in GEPR-9s than in GEPR-3s. In contrast, tanrine concentrations were higher in GEPR-3s than in non-epileptic controls in each region, and in 4 areas were higher in GEPR-3s than in GEPR-9s. Changes resulting from seizure experience consisted of increases in aspartate, glutamate and giycine in seizure-experienced compared to seizure-naive groups in inferior collicuhis and in motor-sensory and frontal cortices. These findings suggest that the high levels of taurine in GEPR-3s and the elevated content of aspartate in GEPR-9s have roles as determinants of seizure severity. The low concentrations of GABA in both types of GEPRs are consistent with a role for this amino acid in determination of seizure susceptibility. Furthermore, the seizure-induced changes in aspartate and glutamate in both types of GEPRs support the concept that these excitatory amino acids mediate changes in seizure predisposition. The current results are in agreement with previous studies indicating that imbalances in neurotransmitter amino acids are important factors in determining seizure behavior in the GEPR. INTRODUCTION As many as 20% of patients with epilepsy cannot be treated successfully with current antiepileptic agents 2°. Thus, the need remains acute for a better understanding of the multffactorial processes underlying epileptogenesis so that more effective treatments can be devised. Results from numerous animal studies have indicated that excessive excitatory amino acid neurotransmission and/or deficient inhibitory amino acid neuronal activity could play a significant role in the inherited or acquired defects responsible for this condition 5'13'21. The genetically epilepsy-prone rat ( G E P R ) is a useful model for studying the involvement of neurotransmitter amino acids in epileptic mechanisms since an imbalance of excitatory and inhibitory amino acid influences appears to be an underlying factor in seizure initiation and/or predisposition in these animals 12. Accordingly, G E P R s have been utilized in neurochemical, electrophysiological and pharmacological studies examining the roles of these transmitters in seizure behavior. Faingnld et al.13 observed a decreased effectiveness of G A B A
iontophoretically appfied into inferior colliculus of GEPR-9s, leading to the suggestion that G A B A e r g i c inhibitory influences may be deficient at this brain site. Furthermore, Millan et al.23 found that microinjection of G A B A antagonists or excitatory amino acid agonists into inferior colliculus conferred audiogenic seizure susceptibility upon normal rats. Conversely, microinjection of G A B A agonists or excitatory amino acid antagonists into the brainstem auditory pathway up to the inferior colliculus blocked audiogenic seizure susceptibility in G E P R - g s 12'14. This latter work also demonstrated the inferior colliculus to be the most sensitive site for pharmacological manipulation. In agreement with this study, Meldrum et al.22 found that microinjection of an excitatory amino acid antagonist into inferior colliculus, substantia nigra or midbrain reticular formation blocked seizures in GEPR-9s with inferior collicular administration being most effective. Lehmann 19 has reported increases in aspartic acid concentrations in 3 of 4 brain regions from severe seizure G E P R s (GEPR-gs) compared to Sprague-Dawley controls, as well as higher potassium-evoked hippocam-
Correspondence: S.M. Lasley, Department of Basic Sciences, University of Illinois College of Medicine at Peoria, Box 1649, Peoria, IL 61656, U.S.A.
64
Audiogenic Seizure Stimulus
pal a s p a r t a t e release in s e i z u r e - p r o n e rats. In contrast, G A B A and t a u r i n e c o n c e n t r a t i o n s w e r e l o w e r in 2 b r a i n
Seizure-Experienced
p----//---
.......
#1 L. . . . . .
#2 :
#3 L ...........
regions of G E P R - 9 s c o m p a r e d to S p r a g u e - D a w l e y controls. O n the o t h e r h a n d , R i b a k et al. 3° o b s e r v e d large increases in G A B A , in the
central
Age, days
I~ Birth
45
52-53
60
76 Sacrifice
t a u r i n e a n d g l u t a m i c acid c o n t e n t
nucleus
o f the
inferior
colliculus o f
G E P R - 9 s r e l a t i v e to S p r a g u e - D a w l e y controls, in addition to increases in cortical t a u r i n e and g l u t a m a t e in the s e i z u r e - p r o n e rats. T h e s e findings c o r r e s p o n d e d to previous i m m u n o c y t o c h e m i c a l d a t a f r o m t h e s e w o r k e r s ' labo r a t o r y indicating i n c r e a s e d n u m b e r s o f G A B A n e u r o n s
Seizure-Naive
~--//
No Seizure Stimuli Fig. 1. Treatment protocol determining seizure-naive and seizureexperienced genetically epilepsy-prone rats. Seizure-inducing sound stimuli were administered once at 3 weekly intervals between the ages of 45 and 60 days of age to constitute seizure-experienced groups. Seizure-naive groups received no sound stimulation, and were classified on the basis of seizure behavior in littermates.
in i n f e r i o r colliculus of G E P R - 9 s c o m p a r e d to S p r a g u e D a w l e y c o n t r o l s 32. H o w e v e r , C h a p m a n et al. 6 r e p o r t e d l o w e r r e g i o n a l c o n t e n t o f a s p a r t a t e and g l u t a m a t e in a few of the 8 b r a i n areas s a m p l e d f r o m G E P R - 9 s r e l a t i v e to S p r a g u e - D a w l e y
controls, b u t f o u n d that aspartic
acid c o n t e n t i n c r e a s e d in i n f e r i o r colliculus of G E P R - 9 s e x p e r i e n c i n g seizures c o m p a r e d to the s a m e t y p e of animals u n d e r resting conditions. T h e G E P R has an i n h e r i t e d susceptibility to seizures i n d u c e d by e x p e r i m e n t e r - c o n t r o l l e d e v e n t s such as sensory stimuli. Originally d e r i v e d f r o m t h e S p r a g u e - D a w ley strain, the G E P R c u r r e n t l y exists as 2 i n d e p e n d e n t l y d e r i v e d colonies, e a c h exhibiting a characteristic convulsive p a t t e r n in r e s p o n s e to an a u d i t o r y stimulus. M e m b e r s o f the s e v e r e seizure c o l o n y ( G E P R - 9 s )
display
s o u n d - i n d u c e d tonic e x t e n s o r c o n v u l s i o n s , while m e m bers o f the m o d e r a t e seizure c o l o n y ( G E P R - 3 s ) display only clonic c o n v u l s i o n s in r e s p o n s e to the acoustical stimulus. S e i z u r e b e h a v i o r in the. G E P R is also r e s p o n -
degree of seizure predisposition. Members of the moderate seizure GEPR-3 colony exhibit a wild running phase which terminates in generalized clonus following a standardized sound stimulus. Members of the severe seizure GEPR-9 colony exhibit a brief wild running phase that terminates in a tonic extensor convulsion. Non-epileptic controls were the offspring of established seizure-resistant control breeders; these animals never exhibited sound-induced seizures. A screening protocol verifies that each animal exhibits an Audiogenic Response Score characteristic of the colony from which it was derived TM. For this study GEPRs were divided into seizure-naive and seizure-experienced groups based on whether a seizure-inducing acoustical stimulus had been presented. Seizure-experienced GEPRs were sound-stimulated once to induce seizures at each of 3 weekly intervals between 45 and 60 days of age, while seizure-naive GEPRs were classified on the basis of seizures observed in littermates (Fig. 1). Experience indicates that use of this latter criterion ensures a very high probability of assignment of the appropriate seizure phenotype. Non-epileptic controls were divided similarly to GEPRs according to the occurrence or absence of exposure to the acoustical stimulus. Lights in the animal rooms were set on a 12:12 cycle commencing at 06.00 h with temperature maintained at 24 + 1 °C.
sive to a n u m b e r o f a n t i e p i l e p t i c drugs useful in tonicclonic and focal types of e p i l e p s y in m a n 27. T h e f o l l o w i n g study was p e r f o r m e d to c o m p a r e seiz u r e - n a i v e and s e i z u r e - e x p e r i e n c e d G E P R s in an a t t e m p t to distinguish e x c i t a t o r y and inhibitory a m i n o acid transmitter changes
underlying
seizure susceptibility f r o m
t h o s e r e l a t e d to seizure e x p e r i e n c e . M o r e o v e r , G E P R - 3 s w e r e i n c l u d e d in the e x p e r i m e n t a l design to d e t e r m i n e w h e t h e r similar a l t e r a t i o n s in t r a n s m i t t e r a m i n o acids w e r e p r e s e n t in b o t h m o d e r a t e and s e v e r e seizure groups. Finally, p r e v i o u s r e p o r t s o f r e g i o n a l b r a i n a m i n o acid c o n c e n t r a t i o n s in G E P R - 9 s h a v e b e e n s o m e w h a t v a r i a n t , particularly with respect to e x c i t a t o r y a m i n o acids and
GABA.
Tissue dissection Age of the animals at sacrifice was 76 _+ 3 days (mean -+ S.D., range: 69-85 days) with generally one animal drawn per litter (n = 6-9 for each seizure-experienced or seizure-naive group). Rats were sacrificed between 10.00 and 13.00 h by decapitation with immediate immersion in liquid nitrogen for 5-6 s. Brains were quickly removed and immediately dissected in a cryostat maintained at -5 °C using the atlas of Paxinos and Watson 26 as a guide. Frontal cortex was removed from the medial half of each hemisphere rostral to the genu of the corpus callosum. Striatum was dissected from a coronal slice bounded by cuts through the head of the eaudate and through the rostral edge of the thalamus. Motorsensory cortex was defined as that cortex overlying the striatum and hippocampus minus dorsomedial (cingulate and occipital) and ventrolateral (pyriform and entorhinal) portions. Thalamus was dissected from a coronal slice bounded by cuts through the rostral edges of this structure and that of the medial geniculate. Hippocampus and inferior collieulus were removed on the basis of observable landmarks. Tissue was stored at -75 °C until analysis.
MATERIALS AND METHODS
Animals Four types of rats were employed in this study. Commercially bred Sprague-Dawley males were obtained from Harlan SpragueDawley, Inc. (Indianapolis, IN) at 65 days of age and utilized as one type of control subject. The other rats were males obtained from 3 independent colonies maintained at the University of Illinois College of Medicine at Peoria. Each colony has been separately derived from Sprague-Dawley stock for a specific type and
Sample preparation and chromatography Amino acids were quantified essentially according to Cohen et al. 8 and Bidlingmeyer et al. 3. Brain regions were homogenized in 20-30 vols of 0.1 M HC1 (constant volume for a given region) and ultrafiltered by centrifugation. Methionine sulfone in 0.1 M HCI was added as an internal standard to an aliquot of each extract, and this mixture was dried under vacuum prior to derivatization with phenylisothioeyanate and a final drying step. Derivatives were redissolved in the initial mobile phase, placed in an automated
65 sample injector and analyzed by binary gradient liquid chromatography with detection by ultraviolet absorbanee at 254 nm (PICOTAG Amino Acid System, Waters Chromatography Div., Milford, MA). Flow rate was 1.0 ml/min. The amino acids of interest - aspartie and glutamie acids, G A B A , taurine and glycine - were quantitated by integration of peak areas with comparison to standards of known concentration included in each run. Tissue pellets from the initial centrifugation were dissolved in 1.0 M NaOH and protein concentrations determined by the method of Smith et al.33 using bovine serum albumin as a standard. Bovine serum albumin, methionine sulfone and amino acid standards were obtained from Sigma Chemical Co. (St. Louis, MO). Mobile phases for the gradient chromatography were obtained directly from Waters Chromatograpy Div. (Milford, MA).
tiates the action of glutamate at an allosteric site on one type of excitatory amino acid receptor, and because its role as an inhibitory transmitter in mammalian forebrain is r e l a t i v e l y m i n o r . T h e m o s t c o n s i s t e n t o b s e r v a t i o n w a s t h a t in 5 o f 6 b r a i n a r e a s a s p a r t i c acid c o n c e n t r a t i o n s w e r e significantly h i g h e r i n G E P R - 9 s c o m p a r e d t o n o n e p i l e p t i c c o n t r o l s in t h e r a n g e o f 7 - 1 2 % , e x c e p t f o r f r o n tal c o r t e x in w h i c h t h e e l e v a t i o n w a s 41%. I n c o n t r a s t , in 2 r e g i o n s in G E P R - 3 s - h i p p o c a m p u s a n d t h a l a m u s a s p a r t i c acid c o n t e n t w a s significantly less t h a n i n n o n epileptic controls. Abnormalities
Data analysis Amino acid concentrations were analyzed by two-factor analyses of variance for each amino acid in each brain region with seizure severity (non-epileptic control, GEPR-3, GEPR-9) and seizure experience as the main factors. Standard t-tests were utilized to determine the bases of significant effects by examining a set of group comparisons established a priori.
in g l u t a m i c acid c o n c e n t r a t i o n s w e r e
less n o t a b l e in G E P R - 9 s c o m p a r e d t o n o n - e p i l e p t i c c o n t r o l s w i t h significant i n c r e a s e s o n l y in h i p p o c a m p u s a n d f r o n t a l c o r t e x . O n t h e o t h e r h a n d , g l u t a m i c acid c o n t e n t in G E P R - 3 s
w a s significantly h i g h e r in f r o n t a l c o r t e x ,
b u t w a s l o w e r in s t r i a t u m c o m p a r e d t o n o n - e p i l e p t i c c o n trols. F e w e r s i g n i f i c a n t c h a n g e s in glycine c o n c e n t r a t i o n s
RESULTS
w e r e p r e s e n t : in G E P R - 9 s g l y c i n e w a s e l e v a t e d in h i p p o c a m p u s a n d d i m i n i s h e d in f r o n t a l c o r t e x r e l a t i v e to c o n -
Aspartate, glutamate, glycine
trois, w h i l e in G E P R - 3 s
glycine w a s l o w e r in s t r i a t u m .
T a b l e I gives t h e r e g i o n a l b r a i n a m i n o acid c o n c e n t r a -
In 3 brain regions (hippocampus, striatum and thala-
t i o n s f o r a s p a r t a t e , g l u t a m a t e a n d glycine a c c o r d i n g t o
m u s ) a s p a r t a t e a n d g l u t a m a t e c o n c e n t r a t i o n s w e r e sig-
s e i z u r e p h e n o t y p e . G l y c i n e is i n c l u d e d b e c a u s e it p o t e n -
n i f i c a n t l y s m a l l e r in G E P R - 3 s t h a n in G E P R - 9 s . A n a l o -
TABLE I
Regional brain excitatory amino acid concentrations by seizure severity Values are expressed as mean ± S.D. in nmol/mg protein with n = 11-17 for each mean. Concentrations from seizure-experienced and seizure-naive animals are combined to show the bases of statistical significance with respect to seizure severity.
Region
Animal
ASP
GL U
GLY 1
Hippocampus
N-E control GEPR-3 GEPR-9
30.5 ± 3.3 27.6 ± 2.8 *'c 34.3 ± 4.7*
113.1 ± 11.8 104.9 ± 10.5 b 124.3 ± 15.1"
11.1 ± 1.4 10.3 ± 0.9 b 12.1 ± 1.5"
Inferior colliculus
N-E control GEPR-3 GEPR-9
32.5 ± 2.7 34.8 -+ 4.7 35.9 ± 3.6**
Motor-sensory cortex
N-E control GEPR-3 GEPR-9
Frontal cortex
68.2 ± 5.4 72.3 ± 7.7 73.1 ± 8.3
18.8 ± 1.0 19.3 ± 1.7 19.9 ± 1.8
46.4 ± 4.0 46.1 ± 4.0 44.1 ± 4.2
114.8 ± 11.0 119.2 ± 11.6 112.9 ± 8.7
8.1 ± 0.8 8.6 ± 0.7 8.2 ± 0.8
N-E control GEPR-3 GEPR-9
39.0 ± 7.1 47.7 ± 12.6 54.9 ± 8.5***
270.3 ± 29.5 314.3 -- 33.0** 296.2 ± 32.1'
Anterior striatum
N-E control GEPR-3 GEPR-9
23.9 ± 2.9 23.9 - 2.3 a 26.6 ± 2.8*
94.3 ± 8.4 84.9 -+ 7.7 **'b 97.5 ± 12.0
8.7 ± 0.7 7.9 ± 1.1 *'a 8.8 ± 0.9
Thalamus
N-E control GEPR-3 GEPR-9
29.2 ± 2.4 24.4 ± 2.9 ***'¢ 31.3 ± 2.9*
73.6 --- 10.6 67.6 ± 6.9 b 77.2 + 8.1
9.5 ± 0.9 9.5 ± 0.9 9.7 - 0.6
ASP, Aspartic acid; GLU, glutamic acid; GLY, glycine. t Giycine is included because it is active at an allosteric site on one subtype of glutamate receptor. ap < 0.05; bp < 0.01; cp < 0.001 compared to GEPR-9 value *P < 0.05; **P < 0.01; ***P < 0.001 compared to the non-epileptic (N-E) control value.
26.3 ± 2.5 26.6 ± 2.1 b 24.4 ± 1.4"
66 gous changes in glycine were noted in 2 areas (Table I). Table II displays the statistically significant changes in aspartate, glutamate and glycine concentrations that occurred as a result of seizures induced by sound stimulation at 3 weekly intervals in G E P R - 3 s and G E P R - 9 s . Increases in content of these amino acids were observed in inferior colliculus of seizure-experienced G E P R - 9 s in comparison to seizure-naive rats of the same seizure phenotype. In addition, elevations in aspartic acid in seizure-experienced G E P R - 9 s were also found in frontal cortex. Changes in these amino acids were noted in seizure-experienced c o m p a r e d to seizure-naive G E P R - 3 s in both cortical areas sampled: glutamate and glycine were increased in motor-sensory cortex as a result of seizure experience while glutamate and aspartate were increased in frontal cortex. With the exception of the change in aspartic acid in frontal cortex (35%), the other increases induced by seizure experience were in the range of 1020%.
In contrast, taurine concentrations were significantly higher in G E P R - 3 s c o m p a r e d to non-epileptic controls in the range of 14-57% in all brain areas sampled. However, taurine content in G E P R - 9 s was significantly higher relative to non-epileptic controls only in hippocampus. Thus, taurine levels in the 2 types of G E P R could be distinguished statistically in 4 brain regions in which values in G E P R - 3 s were higher - inferior colliculus, motor-sensory and frontal cortices and thalamus. Only 2 statistically significant changes in G A B A and taurine concentrations resulted from seizures induced by sound stimulation in G E P R - 3 s and G E P R - 9 s at the 3 weekly intervals. G A B A content in thalamus was higher in seizure-experienced than in seizure-naive G E P R - 9 s , while taurine concentrations in frontal cortex were higher in seizure-experienced than in seizure-naive G E P R - 3 s (data not shown). N o n - e p i l e p t i c c o n t r o l s vs S p r a g u e - D a w l e y
controls
The decreases in G A B A and increases in aspartic acid observed throughout the brains of G E P R - 9 s (Tables I and III) p r e s e n t e d a p p a r e n t discrepancies with previous reports by other laboratories 6"3°'32. Since the experimen-
Taurine, G A B A
Table III gives the regional brain amino acid concentrations for taurine and G A B A according to seizure phenotype. Consistent changes were o b s e r v e d in b o t h these amino acids. G A B A content was significantly lower in both G E P R - 3 s and G E P R - 9 s c o m p a r e d to non-epileptic controls in the range of 12-33% in all brain regions examined. H o w e v e r , there was little differentiation between the 2 types of seizure-prone rats in the magnitude of the decreases in G A B A . G E P R - 3 s and G E P R - 9 s were distinguishable statistically in only 2 brain areas: G E P R - 3 s had significantly lower concentrations of G A B A than G E P R - 9 s in hippocampus, but this relationship was reversed in frontal cortex.
tal subjects (i.e. G E P R - 9 s ) were p r e s u m a b l y identical in each investigation, the selection of control animals for comparison to G E P R s was considered to be one potential differentiating factor. Consequently, 8 commercially b r e d male S p r a g u e - D a w l e y rats were purchased for comparison of regional amino acid concentrations to those in a separate group of 7 non-epileptic controls. The non-epileptic control colony has been b r e d alongside the G E P R colonies to be free of seizure behavior and has been the source of all other control animals utilized in this study.
TABLE II Regional changes in excitatory amino acids resulting from seizure experience
Values are expressed as mean - S.D. in nmol/mg protein ith n = 6-9 for each mean. Region
ASP
GL U
GL Y 1
SN ~ SE
SN ~ SE
SN ~ SE
GEPR-3 GEPR-9
33.7 +- 1.8 --* 38.0 --- 3.6*
-
Motor-sensory cortex
GEPR-3 GEPR-9
-
111.0 -- 7.4 ~ 125.3 --- 10.6" -
8.1 _+ 0.5 ~ -
Frontal cortex
GEPR-3 GEPR-9
40.2 _+ 8.7 ~ 54.4 +- 12.0" 49.4 - 6.7 ~ 59.7 - 7.1"
293.8 - 27.0 --~ 332.2 +- 27.6* -
-
Inferior coiliculus
Animal
68.6 +- 3.5 ~
77.0 --- 9.5*
18.9 -+ 0.9 ~ 20.7 _+ 1.9" 8.9 -+ 0.6*
ASP, Aspartic acid; GLU, glutamic acid; GLY, glycine; SN, mean for seizure-naive animals; SE, mean for seizure-experienced animals; -, no change. i Glycine is included because it is active at an allosteric site on one subtype of glutamate receptor. *P < 0.05 compared to seizure-naive value.
67 TABLE III
d e t e r m i n a t i o n s are s h o w n in T a b l e IV. M o s t n o t a b l e was
Regional brain inhibitory amino acid concentrations by seizure severity
t h e finding that in e a c h b r a i n a r e a s a m p l e d G A B A cont e n t in S p r a g u e - D a w l e y c o n t r o l s was significantly less t h a n that in n o n - e p i l e p t i c controls. T a u r i n e c o n c e n t r a -
Values are expressed as mean -+ S.D. in nmol/mg protein with n = 11-17 for each mean. Concentrations from seizure-experienced and seizure-naive animals are combined to s h o w the bases of statistical significance with respect to seizure severity.
tions w e r e significantly less in s t r i a t u m and i n f e r i o r colliculus o f S p r a g u e - D a w l e y
c o m p a r e d to n o n - e p i l e p t i c
c o n t r o l s , as w e r e a s p a r t a t e , g l u t a m a t e and glycine con-
Region
Animal
TA U
GABA
c e n t r a t i o n s in i n f e r i o r colliculus.
Hippocampus
N-E control GEPR-3 GEPR-9
86.0 -+ 10.4 106.2 - 9.4*** 99.0 _ 11.0"**
19.2 - 2.4 13.8 -+ 1.9 ***'b 16.8 -+ 2.6*
DISCUSSION
Inferior collieulus
N-E control GEPR-3 GEPR-9
33.8 -+ 2.9 53.2 -+ 5.1 ***'c 33.4 - 2.6
25.4 - 3.0 21.9 -+ 3.0*** 21.2 -+ 3.1"**
studies h a v e d e f i n e d an a p p a r e n t i m b a l a n c e o f e x c i t a t o r y
Motor-sensory N-E control cortex GEPR-3 GEPR-9
82.2 -+ 7.6 112.2 _+ 6.9 ***'c 79.0 - 5.9
12.3 - 2.1 9.7 - 1.8"** 9.7 -+ 1.2"**
e v e r , the results o f n e u r o c h e m i c a l investigations h a v e
Frontal cortex
Anterior striatum
Thalamus
N-E control GEPR-3 GEPR-9 N-E control GEPR-3 GEPR-9 N-E control GEPR-3 GEPR-9
Previous
37.5 -+ 3.0 48.7 _ 4.2 ***'c 39.0 -+ 2.9
and
electrophysiological
and i n h i b i t o r y a m i n o acid n e u r o t r a n s m i s s i o n in G E P R brain, particularly in i n f e r i o r colliculus 12-14'22'23. H o w n o t clearly s u p p o r t e d t h e s e findings 6'3°. T h e c u r r e n t study has p r o v i d e d a d d i t i o n a l clarification o f t h e roles of t h e s e
196.1 -+ 24.6 36.2 -+ 3.8 288.5 - 30.4 ***'c 30.7 - 3.0 ***'b 206.5 -+ 11.6 27.4 -+ 3.1'** 118.5 _+ 11.4 134.5 -+ 15.0"* 125.4 -+ 11.5
pharmacological
a m i n o acids by the inclusion o f s e i z u r e - n a i v e as well as s e i z u r e - e x p e r i e n c e d m o d e r a t e a n d s e v e r e seizure G E P R s a l o n g with a c o m p a r i s o n o f 2 types o f c o n t r o l animals.
22.8 -+ 3.1 15.2 _+ 2.0*** 16.1 -+ 3.l***
T h e results suggest that the high c o n c e n t r a t i o n s o f taurine in G E P R - 3 s and the e l e v a t e d c o n t e n t o f aspartic acid in G E P R - 9 s h a v e roles as d e t e r m i n a n t s o f seizure
13.2 - 2.0 10.1 -+ 1.6"** 10.8 -+ 1.4"**
severity. T h e low c o n c e n t r a t i o n s of G A B A in b o t h types of G E P R s indicate a r o l e for this t r a n s m i t t e r in determination
TAU, Taurine; GABA, y-aminobutyric acid. ap < 0.05; bp < 0.01; cp < 0.001 compared to GEPR-9 value. *P < 0.05; **P < 0.01; ***P < 0.001 compared to the nonepileptic (N-E) control value.
of
seizure
susceptibility.
In
addition,
the
c h a n g e s in a s p a r t a t e a n d g l u t a m a t e levels i n d u c e d by seizures in specific b r a i n r e g i o n s in b o t h types o f G E P R s suggest that t h e s e e x c i t a t o r y a m i n o acids m e d i a t e changes resulting f r o m seizure e x p e r i e n c e . Finally, m o d e r a t e seiz u r e G E P R - 3 s h a v e b e e n s h o w n to b e d i f f e r e n t f r o m se-
A n i m a l s o f t h e s e c o n t r o l g r o u p s w e r e sacrificed at
v e r e seizure G E P R - 9 s in t e r m s o f h i g h e r taurine and
ages similar to t h o s e o f the o t h e r animals e m p l o y e d in
l o w e r aspartic and g l u t a m i c acid c o n c e n t r a t i o n s .
this study, and i n f e r i o r colliculus, a n t e r i o r s t r i a t u m and motor-sensory
c o r t e x dissected and p r o c e s s e d
T h e w i d e s p r e a d r e g i o n a l increases in a s p a r t a t e a n d
as de-
d e c r e a s e s in G A B A
scribed in M a t e r i a l s a n d M e t h o d s . T h e results o f t h e s e
o b s e r v e d in G E P R - 9 s in this w o r k
are in g e n e r a l a g r e e m e n t with the changes in tissue
TABLE IV Comparison of regional amino acid content in non-epileptic and Sprague-Dawley controls Values are expressed as mean - S.D. in nmol/mg protein with n = 7-8 for each mean. Region
Animal
ASP
GL U
GLY
Inferior eolliculus
N-E control SD control
31.3 -+ 2.6 26.5 - 2.7*
70.1 - 5.6 61.8 -+ 4.6**
20.8 -+ 1.4 18.1 - 1.2"*
Anterior striatum
N-E control SD control
22.6 -+ 2.9 21.1 - 3.1
92.0 --_ 8.9 93.3 -+ 11.1
9.0 -+ 0.6 8.9 -+ 1.1
Motor-sensory cortex
N-E control SD control
43.2 + 3.9 46.0 + 4.6
116.7 -+ 12.0 128.5 + 9.3
8.2 -+ 1.0 9.4 + 0.5**
TA U 31.6 --- 3.1 24.2 - 1.8"**
GABA 25.1 _+ 2.8 18.6 -+ 1.5"**
122.7 - 11.9 104.0 _+ 8.0**
23.6 -+ 3.2 18.4 _+ 2.1"*
80.3 - 8.6 82.9 -+ 7.0
12.6 + 2.3 10.4 _+ 1.1"
N-E control, non-epileptic control animals; SD control, Spragne-Dawley control animals. See legends of Tables I and III for amino acid abbreviations. *P < 0.05; **P < 0.01; ***P < 0.001 compared to the non-epileptic control value.
68 amino acid levels reported by Lehmann 19. Tissue amino acid concentrations approximate those levels present intracellularly, but tissue aspartate (and glutamate) is undoubtedly distributed between metabolic and transmitter pools. Currently, there are no means to accurately define the contributions to total aspartate from each source, and thus it cannot be determined whether the increases in aspartic acid emanate from one or both sources. Nonetheless, one would anticipate that a general increase in this excitatory amino acid in conjunction with a general decrease in G A B A would promote the initiation and propagation of seizures. In contrast with the increased aspartate content and decreased G A B A concentrations found in inferior colliculus in GEPR-9s in the current study (Tables I and III), Ribak et al. 3° reported significant increases in G A B A , glutamate and taurine in the central nucleus of this structure in severe seizure rats. This apparent discrepancy can be at least partially resolved by consideration of the types of control animals employed. Ribak et al. 30 utilized commercial Sprague-Dawley rats as controls, and by comparison of the data in Tables I, III and IV it is reasonable to conclude that GEPR-9s have significantly greater levels of aspartate, glutamate, glycine (P < 0.05), taurine and G A B A (P < 0.05) relative to Sprague-Dawley controls. Direct statistical comparisons of GEPR-9s and Sprague-Dawley control values were made bearing in mind that these tissues were assayed at different time periods and the means were based on markedly different sample sizes (i.e., 17 and 8, respectively). Other factors may account for differences between the findings of the current investigation and those of Chapman et al. 6. These workers reported elevations in aspartate content in inferior colliculus of GEPR-9s undergoing seizures compared to GEPR-9s at rest, though interictal levels of aspartate and glutamate were lower in some brain areas of GEPR-9s relative to Sprague-Dawley controls. However, these latter amino acid concentrations could have been reduced by close proximity of the last seizure with sacrifice of the animals for analyses. The inferior colliculus is the most important structure in the auditory pathway of the GEPR with respect to audiogenic seizures. It is the site most sensitive to pharmacological manipulations of excitatory amino acid or GABAergic influences 13'14"22. Moreover, infusion of an excitatory amino acid or a G A B A antagonist into inferior colliculus renders a significant proportion of normal rats susceptible to audiogenic seizures 23. A deficiency of neuronal inhibition has also been reported to be present in this structure ~3. It is reasonable then to suggest that the elevated concentrations of aspartic acid (GEPR-9s) and lower amounts of G A B A (GEPR-3s and GEPR-9s) observed in inferior colliculus in this work predispose this site to
seizure initiation and spread as a result of sound stimulation. Concentrations of taurine were higher in all brain regions in GEPR-3s compared to non-epileptic controls as shown in Table III, but changes were generally absent in GEPR-9s. Alterations in taurine content have been reported by others in GEPR-9s 19 but have not been consistently observed across studies. Taurine has been shown to enhance glutamate metabolism in GEPRs 4 and to possess potent anticonvulsant properties when administered intracerebroventricularly or into inferior colliculus in these animals Lr. However, except for specific sites in the brain - such as retina or hippocampal pyramidal cells where taurine appears to act as a transmitter, it serves primarily a neuromodulatory or osmoregulatory role 17. Nonetheless, its anticonvulsant effects suggest that the increased taurine concentrations in GEPR-3s may be an important factor in compensating for the diminished levels of G A B A and thus play a role in determining seizure severity. The most striking results of this study were the statistically significant increases in aspartate, glutamate and glycine concentrations that occurred in inferior colliculus and motor-sensory and frontal cortices in GEPR-3s and/or GEPR-9s (Table II) as a result of the induction of seizures at 3 weekly intervals 16-31 days prior to sacrifice. However, the processes underlying these increases in amino acid content are not apparent. Kindling stimulation in rats causes hippocampal mossy fibers to establish aberrant synaptic contacts (i.e., causes sprouting) even before generalized seizures o c c u r 34. This process is accompanied by increases in kainate receptor binding 29, and presumably glutamate-containing synapses, in the innervated areas. It is known that in GEPRs a lower proportion of animals exhibit their phenotypic seizure behavior on the first exposure to sound stimulation than on the third exposure 24'25"3L, suggesting the presence of a type of kindling. Reigel et al. 2s have shown that this effect of seizure experience occurs independently of the development of seizure susceptibility or severity. Furthermore, Ben-Aft and Gho 2 have reported that brief epileptiform episodes induced in hippocampal slices generate a persistent potentiation of synaptic transmission with characteristics similar to those of long-term potentiation. Presynaptic changes - such as increased aspartate/glutamate release - constitute a major component of the long-term potentiation phenomenon 1"7. Moreover, the findings of Davies et al. x~ in hippocampus suggest that a deficiency in GABAergic inhibition may predispose to this synaptic potentiation. However, the processes of long-term potentiation and sprouting have not been generalized to brain regions such as inferior colliculus and thus may not completely account for these ob-
69 servations. Nonetheless, the increased concentrations of aspartate, glutamate and glyeine induced by seizure experience - especially in the inferior colliculus - would be expected to further promote seizure behavior induced by sound stimulation. Extensive comparisons of regional brain biogenic amine concentrations in GEPR-3s and GEPR-9s have been made previously 9'1°. Deficits in norepinephrine and 5-hydroxytryptamine content were found in both types of G E P R in most areas sampled and were of graded or similar magnitude. The transmitter amino acid profiles in moderate and severe seizure GEPRs in Tables I-III present a different picture, however. While the changes resulting from seizure experience and the decreases in G A B A content were generally of similar magnitude in both types of G E P R , other alterations were more characteristic of one group or the other. Increases in taurine were generally typical of GEPR-3s - in 4 of the 6 brain areas taurine concentrations were significantly greater in these animals than in GEPR-9s. On the other hand, increases in aspartic acid were generally typical of GEPR9s, and in 3 brain regions aspartic and glutamic acid levels were significantly greater in these animals than in GEPR-3s. These findings suggest that aspartate, glutamate and taurine play important roles in distinguishing moderate and severe seizure GEPRs in combination with other reported distinctions between the 2 groups, such as greater elevations in hearing thresholds in GEPR3s 15. Thus, differing patterns of biochemical and physiological changes may underlie the graded seizure severity of these 2 groups. Table V presents a summary of the relative changes observed in transmitter amino acid cOncentrations in this study and relates these findings to qualitative differences in seizure behavior in the G E P R and 2 types of controls involved. Both GEPR-3s and GEPR-9s originated from commercially-bred Sprague-Dawley rats, and it has been
observed that a small proportion of the latter animals exhibit a running response and/or clonic convulsion in the presence of the sound stimulus that induces seizures in GEPRs 27. The non-epileptic controls rarely exhibit seizure behavior, and animals that do are continually excluded from the colony. Non-epileptic controls had the highest levels of G A B A in this investigation coupled with comparatively lower concentrations of aspartate, suggesting a relative dominance of inhibitory amino acid influences commensurate with their inbred seizure-resistant character. In comparison, Sprague-Dawley controls exhibited lower levels of G A B A but similar concentrations of aspartic and glutamic acids to non-epileptic controis except in inferior colliculus where both aspartate and glutamate values were lower. Thus, Sprague-Dawley controls would be expected to be somewhat more susceptible to seizure-inducing stimuli than non-epileptic controls on the bases of these findings. Both types of G E P R possess lower levels of G A B A than non-epileptic controls and demonstrate increases in aspartate and/or glutamate as a result of seizure experience. These changes suggest a relative dominance of excitatory amino acid influences in GEPRs commensurate with their seizure-prone nature. The high amounts of taurine in GEPR-3s and aspartic acid in GEPR-9s may contribute to the distinction between these 2 groups with respect to moderate and severe seizure intensity, respectively. Thus, the results of this work are in general agreement with pharmacological and electrophysiological studies which have suggested that an imbalance of excitatory and inhibitory amino acid influences at least partially accounts for the seizure-prone nature of GEPRs. In conclusion, this investigation has shown that relative to non-epileptic controls the seizure-prone nature of GEPRs is associated with generally low concentrations of G A B A , while seizure severity is differentiated at least partially by abnormalities in the levels of taurine (GEPR-
TABLE V Correlation o f amino acid changes with seizure behavior
See legends of Tables I, III and IV for listing of abbreviations. IC, Inferior collieulus; MSC, motor-sensory cortex; FC, frontal cortex. N-E controls
Amino acid characteristics
Seizure-behavior
SD controls
GEPR-3
GEPR-9
GABA
Highest
< N-E controls
Low
Low
TAU
-
-
H i g h e s t
-
ASP, GLU
-
< N-E controls (IC)
-
Highest ASP
GLU increases with seizure experience (MSC, FC)
ASP, GLU increase with seizure experience (IC, FC)
Seizureprone, moderate intensity
Seizure prone, severe intensity
Non-epileptic
Very low incidence, minimal intensity
70 3s) o r a s p a r t a t e ( G E P R - 9 s ) . O n the o t h e r h a n d , induc-
the functional nature of these n e u r o c h e m i c a l changes.
tion o f m u l t i p l e seizure e p i s o d e s in G E P R s p r o d u c e s regionally-specific increases in a s p a r t a t e ,
glutamate
and
glycine associated with an e n h a n c e m e n t of seizure predisposition. F u r t h e r e x p e r i m e n t a t i o n is necessary to confirm
Acknowledgements. The author wishes to express his gratitude to Mary Green and Robert Burger for excellent technical assistance in the conduct of this work.
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