Neurochemical Research, Vol. 17, No. 9, 1992, pp. 953-959
Involvement of Synaptosomal Neurotransmitter Amino Acids in Audiogenic Seizure-Susceptibility and -Severity of Rb Mice* S. Simler 1,2, L. Ciesielski a,3, J. Clement 1, A. Rastegar 1, and P. MandeP (Accepted December 19, 1991)
The involvement of synaptosomal neurotransmitter amino-acids in seizure susceptibility and seizure severity was explored. The amino-acid contents of brain synaptosomes were determined in three sublines of Rb mice differing in their response to an acoustic stimulus: Rbl, clonic-tonic seizureprone, Rb2, clonic seizure-prone, and Rb3, seizure-resistant. Synaptosomes were prepared from 6 brain areas considered to be involved in seizure activity: olfactory bulbs, amygdala, inferior colliculus, hippocampus, cerebellum, pons-medulla. The steady-state levels of GABA and glycine (Gly), inhibitory amino-acids, of taurine (Tau), an inhibitory neurotransmitter of neuromodulator, of aspartate (Asp) and glutamate (Glu), excitatory amino-acids, as well as of serine (Ser) and glutamine (Gln), two precursors of neurotransrnitter amino-acids, were determined by HPLC. Low levels of Tau, GABA, and Ser in hippocampus, Gly in amygdala, Glu in hippocampus, inferior coIliculus and ports, Gin and Asp in inferior colliculus appeared to correlate with seizure-susceptibility. GABA and Asp in olfactory bulb, Gln in amygda[a, hippocampus and ports, ser in olfactory bulb and pons, appeared to be associated either with seizure-severity or -diversity. A strong involvement of hippocampus (Tau, GABA, Ser, Glu, and Gin) and inferior colliculus (Asp, Glu, Gln) in audiogenic seizure-susceptibility, and of olfactory bulb (GABA, Asp) in seizure-severity and/or -diversity is suggested. KEY WORDS:
Synaptosomes,neurotransmitteramino-acids, audiogenicseizure, seizure-susceptibility,sei-
zure-severity
INTRODUCTION
genetically epilepsy prone rats (7) as well as in three inbred sublines of Rb mice which differ in their responses to an acoustic stimulus (8). The selective breeding of the three Rb sublines began in 1955 (9,10). Common patterns of alterations in amino-acid levels in some brain regions in the clonic-tonic seizure-prone subline Rbl and clonic seizure-prone subline Rb2, as compared with the seizure-resistant Rb3 subline, were suggested as markers of seizure-susceptibility. Inter-subline differences in the steady-state level of neurotransmitter amino-acids between clonic and tonic seizure-prone Rbl and clonic seizure-prone Rb2 seemed to be involved in the modulation of seizure-severity (8). A deficiency
Conflicting results regarding brain neuroactive aminoacids and seizure disorders have been reported (1-5; see 6 for review). The involvement of putative inhibitory and/or excitatory neurotransmitter amino-acids was suggested in seizure-susceptibility and seizure-severity in i Centrede Neurochimiedu CNRS, 5 rue Blaise Pascal, 67084 Strasbourg-Cedex, France. z Charg6ede rechercheINSERM. 3 Address reprint requests to: Dr. L. Ciesielski, Centre de Neurochimie, 5 Rue blaise Pascal, 67084 Strasbourg-Cedex,France. * Special issue dedicatedto Dr. Alan N. Davison. 953
03643190/92/0900-0953506.50/0
9 1992 Plenum Publishing Corporation
954
Simler, Ciesielski, Clement, Rastegar, and Mandel
of Gin and G A B A appeared to correlate with seizuresusceptibility, whereas deficiencies of inhibitory neurotransmitter amino-acids as well as of Asp seemed to be involved in seizure-severity (8,11). The neurotransmitter amino-acids stored in nerve terminals undergo a continuous exchange with the whole cellular amino-acid pool and appear to be directly involved in functional activity (12). A major role of the neurotransmitters present in the synaptosomal fraction is also well documented (6,13-17). It seems therefore of interest to explore the involvement of synaptosomal neurotransmitter amino-acids in seizure-susceptibility and severity. In this regard, we investigated the neurotransmitter amino-acids content of brain synaptosomes in the three sublines of Rb mice differing in their response to an acoustic stimulus. The neurotransmitter amino-acid levels were determined in the synaptosomal fractions of 6 brain regions considered to be involved, in rats (18) and mice (8), in audiogenic seizures manifestations: ponsmedulla, olfactory bulb, amygdala, inferior colliculus, hippocampus, and cerebellum. The distribution of inhibitory and excitatory amino-acids in the corresponding whole brain regions were already investigated in Rb mice and a possible correlation with seizure-susceptibility or -severity delineated (8). Moreover, the involvement of neurotransmitter abnormalities in these 6 brain areas in seizure activity was suggested in genetically epilepsy prone rats (18).
EXPERIMENTAL PROCEDURE
Animals. Swiss albino mice from three different sublines (Rbl, Rb2, Rb3) of the Rb strain were used (8,9,10). Animals were kept under 7 a.m./7 p.m. normal light/dark cycle, with food and water ad libitum. These sublines were supplied by Mrs. A. Lehmann (laboratoire de PhysiologicAecoustique, INRA, Jouy-en-Josas, France) and bred in our laboratory. The mice were submitted at the age of 1 month to an acousticstimulus, in order to confirmtheir geneticallydetermined susceptibility to audiogenic seizures. In response to a 8000 Hz 100 dB acoustic stimulus, Rbl mice developed a clonie-tonic seizure, Rb2 mice developed a clonic seizure while Rb3 mice were seizure-resistant (9,10). Sacrifice and Preparation of Regional Synaptosomal Fractions. Mice were decapitated so that the heads fell directly into 0.9% NaC1 maintained at 0.5~ (15). The heads were moved gently through the fluid to facilitate cooling. The brains were removed from the skulls and 6 brain regions were rapidly and bilaterally dissected on a cold plate: olfactory bulbs, amygdala, inferior eolliculus, hippocampus, cerebellum, and pons-medulla. Synaptosomal fractions were prepared immediately after dissection (19,20). The individual regions of each mouse brain were rapidly homogenized (800 rpm, 10 strokes, 1.5 ml polypropylenecentrifuge tubes with laboratory casted pestles) in 0.4 ml of 0.32 M sucrose (pH 7.0) containing 1 mM 3-mercapto-propionicacid to prevent artifactual increases of GABA during the isolation via inhibition of glutamate
decarboxylase and decrease of GABA release (19,21). Homogenates were centrifuged at 1,000 g for 10 rain to yield the nuclear pellet and the low speed supernatant. The above supernatant was layered onto 1 ml of 1.2 M sucrose in 8 ml polycarbonatetubes and centrifuged in a fixed-angle 50-Ti rotor at 220,000g for 15 min, at a slow acceleration rate in a Beckman L8-55 ultracentrifuge using the tozt facility. The gradient interface was carefully collected with a syringe and diluted with 0.32 M sucrose to a final volume of 2 ml. The suspension obtained was layered onto 1 ml of 0.8 M sucrose and centrifuged as above. The resulting pellet served as synaptosomalpreparation. Amino-Acid Extraction and Analysis. The synaptosomal pellets were suspended in 200 ~1 of 0.1 M perehloric acid (PCA) containing 50 nmol of beta-methyl aspartie acid, used as the internal standard, and then transferred to polypropylene tubes; the polycarbonate tubes were rinsed with 200 ~1 of 0.1 M PCA. The suspension was centrifuged for 15 rain. at 1,000 g at 4~ Amino-acid analysis was performed by high performance liquid chromatographyas ion pairs with dodecyl-sulphate of the underivatized amino-acids according to Seiler and Knodgen (22). Separations were performed on a thermostated (23~ (25 cm x 4.6 mm I.D.) column (Beckman Ultraspher TM I.P.). The eluate flow rate was 1 ml/min (0.2 M ortho-phosphoricacid containing 10 mM sodium dodecyl-sulphate, 0.2 M sodium acetate adjustedto pH 4.5 and containing 10 mM sodium dodecyl-sulphate, methanol, 63%/27%/10% v/v). The eluated material was mixed in a 1/1 ratio with o-phthalaldehyde-2-mercaptoethanolreagent (50 g boric acid and 31.5 g sodium hydroxide in a liter of water, plus 6 ml of a 30% aqueous solution of Brij-35, 3 ml of 2-mercaptoethanol and a solution of 400 mg o-phthalaldehyde dissolved in 5 ml of methanol). This post-column derivatization precludes the problem of the differential stability of the fluorescent o-phthalaldehydederivatives of aminoacids. Moreover, controls with standard solutions were performed assessing the validity of the procedure. Fluorescencewas detected using an Amino Fluoro-monitor, registered and integrated using a SpectraPhysics SP 400 recording integrator. A 100 ~1 loop was used for sample application. The protein content of synaptosomal fraction was measured by the method of Lowry (23). StatisticalAnalysis. Analysis of variance followed by Duncan's test was carried out for significance evaluation of inter-subline differences of steady-state level neurotransmitter amino-acids (N = 6).
RESULTS
Neurotransmitter Amino-Acids in Synaptosomal Fractions. The synaptosomal steady-state level of GABA, taurine, glycine, inhibitory amino-acid neurotransmitters, aspartate and glutamate, excitatory neurotransmitters and of glutamine and serine were determined in the six brain areas listed, in 2 sublines of audiogenic seizureprone mice (Rbl: clonic-tonic, Rb2 clonic) as well as in a seizure-resistant one (Rb3) (Tables I-II). The distribution of synaptosomal versus whole tissue concentration ratio (%) is recorded for Rb3 mice in Figure 1. The significant inter-subline variations (Rbl versus Rb3, Rb2 versus Rb3, Rbl versus Rb2) are recorded in Figure 2. A) Inhibitory Amino-Acids. The levels of the synaptosomal inhibitory amino-acids, vary strongly between the areas examined (Table I). The maximum/minimum
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Involvement of Synaptosomal Neurotransmitter Amino Acids in Audiogenic Seizure-Susceptibility and -Severity of Rb Mice* S. Simler 1,2, L. Ciesielski a,3, J. Clement 1, A. Rastegar 1, and P. MandeP (Accepted December 19, 1991)
The involvement of synaptosomal neurotransmitter amino-acids in seizure susceptibility and seizure severity was explored. The amino-acid contents of brain synaptosomes were determined in three sublines of Rb mice differing in their response to an acoustic stimulus: Rbl, clonic-tonic seizureprone, Rb2, clonic seizure-prone, and Rb3, seizure-resistant. Synaptosomes were prepared from 6 brain areas considered to be involved in seizure activity: olfactory bulbs, amygdala, inferior colliculus, hippocampus, cerebellum, pons-medulla. The steady-state levels of GABA and glycine (Gly), inhibitory amino-acids, of taurine (Tau), an inhibitory neurotransmitter of neuromodulator, of aspartate (Asp) and glutamate (Glu), excitatory amino-acids, as well as of serine (Ser) and glutamine (Gln), two precursors of neurotransrnitter amino-acids, were determined by HPLC. Low levels of Tau, GABA, and Ser in hippocampus, Gly in amygdala, Glu in hippocampus, inferior coIliculus and ports, Gin and Asp in inferior colliculus appeared to correlate with seizure-susceptibility. GABA and Asp in olfactory bulb, Gln in amygda[a, hippocampus and ports, ser in olfactory bulb and pons, appeared to be associated either with seizure-severity or -diversity. A strong involvement of hippocampus (Tau, GABA, Ser, Glu, and Gin) and inferior colliculus (Asp, Glu, Gln) in audiogenic seizure-susceptibility, and of olfactory bulb (GABA, Asp) in seizure-severity and/or -diversity is suggested. KEY WORDS:
Synaptosomes,neurotransmitteramino-acids, audiogenicseizure, seizure-susceptibility,sei-
zure-severity
INTRODUCTION
genetically epilepsy prone rats (7) as well as in three inbred sublines of Rb mice which differ in their responses to an acoustic stimulus (8). The selective breeding of the three Rb sublines began in 1955 (9,10). Common patterns of alterations in amino-acid levels in some brain regions in the clonic-tonic seizure-prone subline Rbl and clonic seizure-prone subline Rb2, as compared with the seizure-resistant Rb3 subline, were suggested as markers of seizure-susceptibility. Inter-subline differences in the steady-state level of neurotransmitter amino-acids between clonic and tonic seizure-prone Rbl and clonic seizure-prone Rb2 seemed to be involved in the modulation of seizure-severity (8). A deficiency
Conflicting results regarding brain neuroactive aminoacids and seizure disorders have been reported (1-5; see 6 for review). The involvement of putative inhibitory and/or excitatory neurotransmitter amino-acids was suggested in seizure-susceptibility and seizure-severity in i Centrede Neurochimiedu CNRS, 5 rue Blaise Pascal, 67084 Strasbourg-Cedex, France. z Charg6ede rechercheINSERM. 3 Address reprint requests to: Dr. L. Ciesielski, Centre de Neurochimie, 5 Rue blaise Pascal, 67084 Strasbourg-Cedex,France. * Special issue dedicatedto Dr. Alan N. Davison. 953
03643190/92/0900-0953506.50/0
9 1992 Plenum Publishing Corporation
954
Simler, Ciesielski, Clement, Rastegar, and Mandel
of Gin and G A B A appeared to correlate with seizuresusceptibility, whereas deficiencies of inhibitory neurotransmitter amino-acids as well as of Asp seemed to be involved in seizure-severity (8,11). The neurotransmitter amino-acids stored in nerve terminals undergo a continuous exchange with the whole cellular amino-acid pool and appear to be directly involved in functional activity (12). A major role of the neurotransmitters present in the synaptosomal fraction is also well documented (6,13-17). It seems therefore of interest to explore the involvement of synaptosomal neurotransmitter amino-acids in seizure-susceptibility and severity. In this regard, we investigated the neurotransmitter amino-acids content of brain synaptosomes in the three sublines of Rb mice differing in their response to an acoustic stimulus. The neurotransmitter amino-acid levels were determined in the synaptosomal fractions of 6 brain regions considered to be involved, in rats (18) and mice (8), in audiogenic seizures manifestations: ponsmedulla, olfactory bulb, amygdala, inferior colliculus, hippocampus, and cerebellum. The distribution of inhibitory and excitatory amino-acids in the corresponding whole brain regions were already investigated in Rb mice and a possible correlation with seizure-susceptibility or -severity delineated (8). Moreover, the involvement of neurotransmitter abnormalities in these 6 brain areas in seizure activity was suggested in genetically epilepsy prone rats (18).
EXPERIMENTAL PROCEDURE
Animals. Swiss albino mice from three different sublines (Rbl, Rb2, Rb3) of the Rb strain were used (8,9,10). Animals were kept under 7 a.m./7 p.m. normal light/dark cycle, with food and water ad libitum. These sublines were supplied by Mrs. A. Lehmann (laboratoire de PhysiologicAecoustique, INRA, Jouy-en-Josas, France) and bred in our laboratory. The mice were submitted at the age of 1 month to an acousticstimulus, in order to confirmtheir geneticallydetermined susceptibility to audiogenic seizures. In response to a 8000 Hz 100 dB acoustic stimulus, Rbl mice developed a clonie-tonic seizure, Rb2 mice developed a clonic seizure while Rb3 mice were seizure-resistant (9,10). Sacrifice and Preparation of Regional Synaptosomal Fractions. Mice were decapitated so that the heads fell directly into 0.9% NaC1 maintained at 0.5~ (15). The heads were moved gently through the fluid to facilitate cooling. The brains were removed from the skulls and 6 brain regions were rapidly and bilaterally dissected on a cold plate: olfactory bulbs, amygdala, inferior eolliculus, hippocampus, cerebellum, and pons-medulla. Synaptosomal fractions were prepared immediately after dissection (19,20). The individual regions of each mouse brain were rapidly homogenized (800 rpm, 10 strokes, 1.5 ml polypropylenecentrifuge tubes with laboratory casted pestles) in 0.4 ml of 0.32 M sucrose (pH 7.0) containing 1 mM 3-mercapto-propionicacid to prevent artifactual increases of GABA during the isolation via inhibition of glutamate
decarboxylase and decrease of GABA release (19,21). Homogenates were centrifuged at 1,000 g for 10 rain to yield the nuclear pellet and the low speed supernatant. The above supernatant was layered onto 1 ml of 1.2 M sucrose in 8 ml polycarbonatetubes and centrifuged in a fixed-angle 50-Ti rotor at 220,000g for 15 min, at a slow acceleration rate in a Beckman L8-55 ultracentrifuge using the tozt facility. The gradient interface was carefully collected with a syringe and diluted with 0.32 M sucrose to a final volume of 2 ml. The suspension obtained was layered onto 1 ml of 0.8 M sucrose and centrifuged as above. The resulting pellet served as synaptosomalpreparation. Amino-Acid Extraction and Analysis. The synaptosomal pellets were suspended in 200 ~1 of 0.1 M perehloric acid (PCA) containing 50 nmol of beta-methyl aspartie acid, used as the internal standard, and then transferred to polypropylene tubes; the polycarbonate tubes were rinsed with 200 ~1 of 0.1 M PCA. The suspension was centrifuged for 15 rain. at 1,000 g at 4~ Amino-acid analysis was performed by high performance liquid chromatographyas ion pairs with dodecyl-sulphate of the underivatized amino-acids according to Seiler and Knodgen (22). Separations were performed on a thermostated (23~ (25 cm x 4.6 mm I.D.) column (Beckman Ultraspher TM I.P.). The eluate flow rate was 1 ml/min (0.2 M ortho-phosphoricacid containing 10 mM sodium dodecyl-sulphate, 0.2 M sodium acetate adjustedto pH 4.5 and containing 10 mM sodium dodecyl-sulphate, methanol, 63%/27%/10% v/v). The eluated material was mixed in a 1/1 ratio with o-phthalaldehyde-2-mercaptoethanolreagent (50 g boric acid and 31.5 g sodium hydroxide in a liter of water, plus 6 ml of a 30% aqueous solution of Brij-35, 3 ml of 2-mercaptoethanol and a solution of 400 mg o-phthalaldehyde dissolved in 5 ml of methanol). This post-column derivatization precludes the problem of the differential stability of the fluorescent o-phthalaldehydederivatives of aminoacids. Moreover, controls with standard solutions were performed assessing the validity of the procedure. Fluorescencewas detected using an Amino Fluoro-monitor, registered and integrated using a SpectraPhysics SP 400 recording integrator. A 100 ~1 loop was used for sample application. The protein content of synaptosomal fraction was measured by the method of Lowry (23). StatisticalAnalysis. Analysis of variance followed by Duncan's test was carried out for significance evaluation of inter-subline differences of steady-state level neurotransmitter amino-acids (N = 6).
RESULTS
Neurotransmitter Amino-Acids in Synaptosomal Fractions. The synaptosomal steady-state level of GABA, taurine, glycine, inhibitory amino-acid neurotransmitters, aspartate and glutamate, excitatory neurotransmitters and of glutamine and serine were determined in the six brain areas listed, in 2 sublines of audiogenic seizureprone mice (Rbl: clonic-tonic, Rb2 clonic) as well as in a seizure-resistant one (Rb3) (Tables I-II). The distribution of synaptosomal versus whole tissue concentration ratio (%) is recorded for Rb3 mice in Figure 1. The significant inter-subline variations (Rbl versus Rb3, Rb2 versus Rb3, Rbl versus Rb2) are recorded in Figure 2. A) Inhibitory Amino-Acids. The levels of the synaptosomal inhibitory amino-acids, vary strongly between the areas examined (Table I). The maximum/minimum
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