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In vivo release of neuroactive amines and amino acids from the hippocampus of seizure-resistant and seizure-susceptible rats

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In vivo release of neuroactive amines and amino acids from the hippocampus of seizure-resistant and seizure-susceptible rats
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  Neurochem. Int. Vol. 8, No. 4, pp. 513-520, 1986 0197-0186/86 $3.00 +0.00 Printed in Great Britain Pergamon Journals Ltd N V VO RELEASE OF NEUROACTIVE AMINES AND AMINO ACIDS FROM THE HIPPOCAMPUS OF SEIZURE-RESISTANT AND SEIZURE-SUSCEPTIBLE RATS ANDERS LEHMANN*, MATS SANDBERG'I and RYAN J. HUXTABLE* *Department of Pharmacology, University of Arizona Health Sciences Center, Tucson, AZ 85724, U.S.A. tInstitute of Neurobiology, University of G6teborg, G6teborg, Sweden (Received 24 July 1985; accepted l0 October 1985) Abstract--The & vivo release of six radiolabeled amino acids or amines in the hippocampus was investigated in genetically seizure-resistant and seizure-susceptible rats. No differences were found in the spontaneous or high K+-evoked efflux of 7-aminobutyric acid, norepinephrine or its metabolites, taurine or ~-aminoisobutyrate. However, seizure-susceptible rats released significantly more D-aspartic acid on K+-depolarization. The initial rate of release (flux) was also higher. In view of the ubiquity of synapses in the mammalian brain utilizing excitatory amino acids as their transmitter, we propose that an exaggerated release of these excitants might be a determinant of the innate seizure susceptibility in these animals. INTRODUCTION The genetically seizure-susceptible (SS) rat has been established as a useful model in the study of the neurochemistry of epilepsy (Huxtable, 198 ; Bonhaus et al., 1983). The SS rat has, throughout its life, a consistently diminished threshold for electroshock and chemically-induced seizures. Loud noises also precipitate seizures in the SS rat in a predictable fashion. The SS rat exhibits a number of differences from the seizure-resistant (SR) animal. The brain shows a lower rate of transport of the neuroinhibitory sub- stance, taurine (Bonhaus and Huxtable, 1983), and the synaptosomal content of endogenous taurine and phosphoethanolamine is lower (Bonhaus et al., 1984). There are, furthermore, abnormalities in biogenic amine content, with the levels of norepinephrine being lower in most brain areas, and serotonin being decreased in the cerebral hemispheres and the mid- brain (Jobe et al., 1973, 1982; Jobe and Laird, 1981). There also seem to be some differences in the glutamate-GABA relationship, in that taurine, in vitro, stimulates glutamic acid decarboxylase activity in homogenates of SS brain, but is without effect on decarboxylase activity in SR brains (Bonhaus and Huxtable, 1983). Furthermore, there are suggestions that glutamate turnover is lower in SS brains (Bonhaus and Huxtable, 1985). Abbrev&tions: GABA, ?-aminobutyric acid; SR, seizure resistant; SS, seizure susceptible. It is uncertain how any of these abnormalities relate to the SS state. However, the monoaminergic deficiency has been postulated to be one factor contributing to seizure susceptibility (Jobe and Laird, 1981). It is unlikely that any one abnormality can be pinpointed as the determinant of seizure susceptibility, a condition which most probably is of multifactorial srcin. In the work that has been reported to date, the neuroexcitatory amino acids have been ignored. In this report, we compare the spontaneous and high K+-induced release of radiolabeled excitatory and inhibitory neurotransmitters or neuromodulators from the hippocampus of SR and SS rats in vivo. EXPERIMENTAL PROCEDURES Animals Rats (250 350g) of either sex were used. SR Sprague Dawley rats were obtained from the Division of Animal Resources, University of Arizona. Genetically SS Spragu~ Dawley rats of the UAZ-AGS (SD) strain (Consroe et al., 1981) were obtained from the University of Illinois at Peoria. All SS rats had an audiogenic response score of 9 (maximal responders) as defined by Jobe et al. (1973). Chemicals [1-14C]~-Aminoisobutyric acid (1.68 TBq/mol) was pur- chased from ICN, Chemical and Radioisotope Division, Irvine, Calif. D-[2,3-3H]Aspartic acid (518TBq/mol) and e-[7-~H(N)]norepinephrine (855TBq/mol) were obtained from New England Nuclear (Boston, Mass., U.S.A.). [u-laC]Taurine (4.18 TBq/mol) and [2,3JH]GABA (2890 TBq/mol) were from Amersham International, Arlington Heights, Ill. ?-Vinyl-GABA was from Centre 513  514 ANI)ERS LEHMANN et al. Slope = y6 (min-1) /i \\ B. A. LOg e Aa (cpm .rain- 1) / ~ \ \k Slope = - y (min - ') ~(~ (min - ') Ar~eund~ (cpm) Area under %% -- curve = A (cpm) X Time Time Fig. I. Sketch of curve-fitting equations. Each term of F L represents a semilogarithmic curve of the form of Fig. 1A; F~ represents a curve of the form of Fig. lB. The combined equation F~ + F, approximates a curve of the form shown, for example, in Fig. 3. de Recherche Merell International (Strasbourg, France). Radiochemicals were used at the purchased specific activities. Brain dialysis Rats were anesthetized with 0.2 ml/kg Innovar-Vet" i.p. (0.4rag fentanyl/ml and 20rag droperidol/ml). Atropine sulfate (0.2mg/kg) was administered hourly to prevent bradycardia. The anesthetic depth was estimated inter- mittently by checking the corneal reflex. When necessary, a supportive dose of Innovar-Vet e was given. Typically, 0.6 ml/kg was administered during the entire course of an experiment. The body temperature was kept at 37.5 + 0.5C by means of a heating plate. The rats were placed in a Kopf stereotaxic frame with the incisor bar set at -3.3 mm A dialysis tubing (o.d. 0.3 mm) was implanted in the right hippocampus at the coordinates P 4.8, L 5.0 and V 7.5 according to the atlas of Paxinos and Watson (1982). The design of the dialysis probe is described by Sandberg et al. (1986). The length of the tubing was 3 mm and the molecular weight cutoff was 3000 dalton. The fiber was perfused for 40min at 2.6/~l/min with either: (a) [14C]taurine (1.7 GBq/I) plus D-[3H]aspartic acid (2.8 GBq/I); or (b) [14C]e-aminoisobutyric acid plus [3H]- norepinephrine (2.8 GBq/I). In one set of experiments, the fiber was perfused with [3H]GABA (3.7 GBq/I) and 10 mM 7-vinyl-GABA. Immediately after the preloading, the dialysis fiber was perfused (2.6 l~l/min) with Krebs-Ringer bicarbonate solution (pH 7.4) of the following composition (mM): NaCI 122.0; KCI 3.0; MgSO 4 1.2; KHzPO 4 0.4: NaHCO 3 25, CaC12 1.2. Perfusate was collected in 10-min fractions for 120 rain. At this time the buffer was replaced by modified Krebs Ringer bicarbonate medium containing 100 mM K + with a corresponding reduction of Na +. The high K+-solution was perfused for 20 rain, and thereafter the srcinal Krebs Ringer bicarbonate buffer was reintro- duced for 60 rain. Aliquots of the dialysates (20~1) were mixed with 5 ml scintillation fluid (Aqueous Counting Scin- tillant, Amersham) and '4C-/:~H-radioactivity was measured in a Beckman LS 1800 scintillation counter. In the experi- ments with norepinephrine and aminoisobutyrate, the per- fusate was collected in 10/~1 1,4-dithiothreitol (10 mg/ml) to prevent oxidation of norepinephrine. An aliquot (5,ul) of the perfusate was used for determination of total counts and 20~1 was applied to an AG50W-X8 column (H+-form, 12 x 0.5cm) in order to separate norepinephrine and its nonbasic metabolites. Double-deionized water was added (4 ml) and the last 2 ml (containing metabolites) was col- lected and counted. All values were expressed as cpm/20/~1 perfusate. Data analysis Washout data were fitted to the equations F~ = A:~e ~' + Bile /' and C6 b~= - e :~([-e "'). 7(7 +,5) Using the MLAB curve-fitting program on a DEC-20 computer. For the washout period 0-120 min, data were fitted to F~, and for periods thereafter to F~ + F z. In these equations, :~, fl, 7 and 6 are rate constants (mint); A, B and C are compartment sizes (tool); and Act, Bfl and C6/7(7 + 3) are initial fluxes from these compart- ments (mol.min ~). The significance of these terms is clarified in Fig. 1. Function F~, a biexponential etttux curve, approximates the nondepolarizing efflux of radioactivity from the brain during the washout phase. Function F 2 approximates the stimulated efllux seen on K+-depolarization. To give physical significance to these compartments, A approximates the extracellular pool, "B" the nonneuronal intracellular pool and "C" the neuronal pool. For pool A. in particular, this approximation is limited by the rate of movement between A and B for the substance. The greater  Amino acid release and seizure susceptibility 515 Table 1. Kinetic constants for [14C]taurine and [t4C]ct-aminoisobutyric acid in SR and SS rats [~4C]Taurine SR SS [14C]ct-Aminoisobutyric acid SR SS Rate constants (min ') ct 0.14 _+ 0.11 0.14 _+ 0.06 0.07 _+ 0. I 0 0.05 + 0.07 0.01 -+ 0.00 0.01 -+ 0.00 0.004 -+ 0.004 0.007 -+ 0.006 7 0.04 _+ 0.02 0.04 _+ 0.01 -- -- fi 0.08 -+ 0.06 0.39 -+ 0.33* -- -- Relative compartment sizes A 100 -+ 105 66 _+ 29 100 -+ 104 93 _+ 72 B 46 -+ 19 63 -+ 16 159 _+ 77 359 _+ 604 C 15-+12 16-+7 -- -- Relative fluxes (min i) Act 100 -+ 130 48 -+ 38 100 -+ 179 54 -+ 103 B~ 2-+2 4-+2 4-+4 6-+4 C6(~, +6)1~ 4-+3 5_+6 -- -- The emux data for [14C]taurine were fitted to F t + F 2. The data for [l~]ct-aminoisobutyric acid were fitted to FI only (no fit obtainable to F2). Acre -~' and Bile -at were fitted separately for one experiment in the SS group. The values are mean _+ SD. SR group: n = 5, [~4C]taurine; n = 4, [14C]ct-aminoisobutyric acid. SS group: n = 6. *0.05 < P < 0.10 (Student's t-test, unpaired). the rate of backflux, the more will pool A exaggerate the apparent size of the extracellular pool. For convenience in comparing data for the different substances, pool sizes and fluxes are reported on Tables 1-3 as percentages of pool A. As the specific activities used differed, and as the specific activities of some substances would be affected by endogenous concentrations, tabulation of actual counts (shown in the figures) would not permit such comparisons. RESU LTS Fiber dialysis Dialysis of a brain region through an implanted semipermeable fiber allows continuous sampling of extracellular fluid for its dialyzable constituents (Hamberger et al., 1983). All of the experiments we performed involved the washout of preloaded radio- active substances from the brain under both depolar- izing and nondepolarizing conditions. The curve- fitting model used on the washout data permits the derivation of seven constants (and three derived constants) from a limited number of data points. The standard deviations calculated for these constants are high because many of the parameters are strongly influenced by one or two data points. For this reason, this method underestimates degrees of significance. Table 2. Kinetic constants for [3H]norepinephrine and its metabolites in SR and SS rats [3H]Norepinephrine [3H]Norepinephrine metabolites SR SS SR SS Rate constants (min i) ct 0.15 + 0.18 0.13 + 0.11 0.10 _+ 0.05 0.09 + 0.12 fl 0.005 _+ 0.004 0.010 _+ 0.004 0.009 + 0.004 0.012 + 0.003 0.07 _+ 0.05 0. I 1 -+ 0.10 0.08 + 0.03 0.06 + 0.03 0.50 -+ 0.32 0.36 + 0.22 0.58 + 0.34 0.39 + 0.58 Relative compartment sizes A 100+ 129 91 + 148 100+51 222_+224 B 34 + 18 32 + 3 168 _+ 105 179_+ 94 C 8_+6 5_+2 23_+21 17_+2 Relative fluxes (min i) Act 100 + 185 16 _+ 30 100 _+ 83 350 _+ 630 Bfl 0_+0 0_+0 12+ 10 10_+8 C6(~ +6)/y I _+ I I + 1 13 +9 13 -+ 10 The ettlux data for [3H]norepinephrine and 3H-labeled metabolites were fitted to F, + F 2. There were no significant differences (Student's t-test) between SR and SS with respect to rate constants and compartment sizes for [3H]norepinephrine or 3H-labeled metabolites of norepinephrine. The values are mean + SD for 5 animals/group. In some cases, specific constants could not be fitted.  516 ANDERS LEHMANN cl ~//'. Table 3. Kinetic constants for D-[~H]aspartic acid and [3H]GABA in SR and SS rats D-[3H]Asparlic acid [3H]GABA SR SS SR SS Rate constants (rain ~) 0.06 ± 0.07 0.17 + 0.06* 0.20 :_ 0.15 (). 15 ~ 0. I I fl 0.005 + 0.004 0.006 _+ 0.002 0.005 + 0.002 0.005 + 0.001 7 0.10 + 0.04 0.13 + 0.02 0.14 + 0.01 0.14 + 0.02 6 0.25 + 0.30 0.03 _+ 0.01 0.25 + 0.30 0.03 + 0.00 Relative compartment sizes A 100 + 65 145 + 147 100 ~ 163 90 ~ 153 B 149+98 235_+42 76L25 74+ 12 C 35 + IS 75 + 22** 21 + 10 19 ~ 7 Relative fluxes (rain ') A:t 100 + 134 522 + 546 100 + 181 75 + 153 Bfl 21 +10 27+4 I +0 I 60 C6(7 + 6):7 173 + 116 1021 + 413 ** 33 + 26 47 + 20 The efflux data for D-[3H]aspartic acid and [3H]GABA were fitted to F~ + F~. The values represent mean _+ SD for 5 animals/group. In some cases, specific constants could not be fitted. *0.015 < P < 0.05:**0.010 < P < 0.25: ***0.00l < P < 0.005. However, the curve-fitting provides the most efficient way of extracting the maximum amount of informa- tion from a limited data array. Perfusion of the dialysis fiber with saline resulted in a biexponential decrease in radioactivity in the dialysate for all compounds (Figs 2 4). Introduction of 100raM K + in the perfusion fluid resulted in stimulated release of radioactivity for all substances except ct-aminoisobutyric acid. Taurine The greater rate constant for the upswing of the K +-depolarization peak approached significance for the SS as compared to the SR group (Table 1: Fig. 2A). ~-Aminoisobutyric acid a-Aminoisobutyric acid is a nonmetabolizable amino acid which is transported by the neutral amino acid transport system. This substance, with no known neuroactivity, was included as a control. Washout kinetics obeyed a simple biexponential curve, K +- depolarization having no effect on release (Table I; Fig. 2B). Norepinephrine The hippocampus has sparse noradrenergic innervation. The main finding of interest with the norepinephrine experiment was the extremely low fluxes from compartments B and C (Table 2; Fig. 3A). A greater proportion of the nonbasic metabolites of norepinephrine, on the other hand, were contained within compartments B and C, and their flux rates were higher (Table 2; Fig. 3B). D-Aspartic acid D-Aspartate, a nonendogenous analog of gluta- mate and L-aspartate, is an excitatory amino acid. K+-stimulated release of D-aspartate was signifi- cantly higher from SS as compared to SR rats (Figs 4A). Statistical comparison shows both a higher compartment size in the K +-releasable pool, compartment C, and a faster initial flux from this compartment (Table 3). In addition, the rate constant was also higher. GA BA In order to study the washout of GABA from the hippocampus, it was necessary to inhibit the degrading enzyme, GABA transaminase. This was achieved by inclusion of 7-vinyl-GABA in the per- fusate during the loading (Lippert et al., 1977). As can be seen in Fig. 3B, the washout curves from SS and SR brains were almost superimposable. None of the calculated parameters differed between the two groups. DISCUSSION The use of implanted semipermeable fibers is a powerful technique that permits sampling of substances in extracellular fluid, without changes in extracellular osmolarity, volume or ionic strength (Lehmann et al., 1983, 1985). This technique has not been applied before to the brains of epileptic animals. One limitation of the procedure is that, due to the delicacy of the fibers, the animal is maintained  Amino acid release and seizure susceptibility 517 1500 5000- A 1000 3000 i 500 1000 2O0 100 0 40 8'0 1~)0 160 200 300 Time (min) o Fig. 2A. In vivo release of [~4C]taurine from the rat hippo- campus. A dialysis probe was implanted into the right hippocampus of SR (O) and SS (0) rats. The fiber was perfused for 40min at 2.6pl/min with 1.7GBq/1 [~4C]- taurine. The Krebs-Ringer bicarbonate buffer was perfused for 120min followed by perfusion with 100mM K ÷- containing saline; 20 min later, normal saline was reintro- duced for 60 min. Ten-minute fractions of perfusate were collected during the entire course of the experiment and the radioactivity was determined. The curves are means of 5 experiments for each group. mini A 4 0 00 120 160 2()0 Time(min) Fig. 3A. In vwo release of [3H]norepinephrine from the rat hippocampus. Conditions are as for Fig. 1 except that [3H]norepinephrine was preloaded at 2.8 GBq/I. [3H]Nor- epinephrine in the dialysate was separated from [3H]metab- olites on a cation-exchange column. SR rats (O); SS rats (O). The curves are means of 5 experiments for each group. 1000 B °° 500- i 400- 20 ~ m 200- 100~ ~ 12 1 0 100- Time (min) Fig. 2B. In vivo liberation of [J4C]ct-aminoisobutyric acid from the hippocampus of SR (O) and SS (0) rats. The perfusion scheme was identical to that described in Fig. 2A except that the [~4C]~-aminoisobutyric acid was preloaded at 1.4 GBq/I. The curves are means of 5 (SR) or 6 experi- ments (SS). aim B 70 0 40 8'0 120 160 200 Time (min) Fig. 3B. In vivo release of [~H]metabolites of [3H]norepi- nephrine. Conditions are as for Fig. 3A. SR rats (O); SS rats (0). The curves are means of 5 experiments for each group.
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