REDUCTION OF E~DO~~NOUS LEVEL, UPTAKE AND RELEASE OF TAURINE AFTER rNTRASTRIATAL KAXNIC ACID INJECTION P, PLACHETA’, E, SINGER’, G. SC~I@JEWK~, K. IV~ECKL~and M. KAROBATE? “Institute of Fh~rn~~~o~, University of Vienna, Austria and zDepartment of ~x~r~rn~ntal Psychiatry, Psychiatrische Univars~t~tsklinik, University af Vienna, Austria (Accepted 17 October 1978)
Summary-Rats
received single unilateral stereotaxic injections of 2gg of kainic acid into the let? caudate nucfeus 10 days before sacrifices On the lesioned side, a ~~~j~~~t reduction of e~oge~o~ taurine levels and high affinity uptake of’ 3H-taurine into crude synaptosoma~ (Pa) fractions to about 75% of control vames was found. In agreement with previous reports on the nemotoxic action of uptake into P,-fractions kainic acid, a marked reduction of ~ndogeuons GABA levels and ‘%XABA was observed, while %-dapamine uptake was not sign~fi~~t~y changed. In superfusion experiments with prefabeled P2-fractions prepared from lesioned striata, K *(MmM)-induced release af “H-taurine and “*C-GABA, but not of 3H-dopamine, was reduced. The results are compatible with the hypothesis that taurine in the striatum of the rat is partially localized in interneurones.
Taurine, which is present in the brain in high concentrations, exhibits properties which indicate a possible role as neurotra~m~tter or ne~rom~ulator (for review see: Huxtable and Barbeau, 1976; Collins, 1977). It exhibits a depressant effect on neuronat activity of the cortex and brainstem of the cat (Curtis and Watkins, 1973) and it is released from the cerebral cortex on electrical stimnla?ion (Jasper and Koyama, 1969). High affinity uptake (S&mid, Sieghart and Karobath, 1975) and ~t~si~m-evoke release of taurine have been shown in rat cerebral cortex slices and synaptosomes (Kaczmarek and Davison, 1972; Sieghart and Heck], 1976; Placheta and Singer, 1977). However, these studies as well as electron microscopic autoradiography (Ehinger, 1973) or subcellular fractionation techniques with tissue cultures from glioma or neurobl~toma ceils (Sscbrier and Thompson, 1974; Sieghart and KarobatB 1976) do not ~oncl~siv~~y point to a sole neuronal localization of taurine. Recently, it has been suggested that microinjections of kainic acid inta the corpus striatum of the rat cause a selective degeneration of cholinergic and gabaergic neurones intrinsic to this region (Coyle and Schwartz, 19%; McGeer and McGeer, 1976). Therefore, if a ~onsiderabIe portion of taurine is present in striatd interneurons, Iesions with kainic acid also should lead to changes of nenro~b~rni~i parameters of &urine. In the present study, endogenous levels, high affinity uptake and release of taurine were studied using crude synaptosomal fractions derived from rat striata after intrastriatal injections of kainic acid. Key words: striatum, kainic acid, taurine, GA&A, dopamine. Abbreviations: GABA = 4-ami~o-n-b~t~jc acid, DA = dopamine.
Adult Sprague-Dawley rats of either sex were anesthetized with 50mgilcg (i.p.) ~ntobarbital. Two pg of kainic acid dissolved in 1 ,ul of buffered isotonic saline were injected stereotaxically into the head of the left caudate nucleus (coordinates: AP + 8.38; L + 2.8; DV -t 0.2; according to K&rig and Klippel, 1967), using a 0.3mm Hamilton cannnla. After 10 days the animals were decapitated and the striata of the injected and contralateral intact (control) sides were dissected on a chilled glassplate and homogenized in 10 vol (w/v) of buffered (2mM Tris-HCl, pH 7.2) icecold 0.32 M sucrose. Crude synaptosomal (Pa)fractions were prepared by conventional differentiai ~~trjfugation techniques (Whittier, 1965).
The PZ-fractions were resuspended in a medium containing in mM: NaCI, 128; KCI, 5; MgCI,, 1.2; CaCla, 0.5; d-glucose, 10; sucrose, 10; Tris-HCI, 15 (pH 7.4). Incubations with “C-GABA were performed in the presence of lO$vf aminooxyacetic acid to inhibit GABA catabolism, In the exper~ents with 3H-DA, ascorbic acid fl.14mM) and a rnono~i~ oxidase inhibitor, nialamide, (12.5pM) were included in the uptake medium. For uptake experiments aliquots of the resuspended P,-fractions, equivalent to approximately 3-6mg of original tissue wet weight, were preincubated for 5 min at 37°C in a shaking water bath under air. Then, the labeled compounds were added in 20 ,~l of medium to give a final concerttration of SpM 3H-taurine, 2.7&f ‘%X%ABA, or 0.05 yM “H-DA, respectively, (gnat volume 1.0ml) and the ~n~ubatjons were continued for another 10 min. ~~~nbations were stopped by immediate filtra-
399
400
P. PLACHETA,E.
SINGER,
G. SCH~~NJJECK, K. HECKLand M. KAROBATH
tion through cellulose nitrate filters (Schleicher and Schiill, 0.45pm pore size). The filters were rinsed 3 times with 4ml of medium at room temperature and radioactivity was determined by liquid scintillation counting. Uptake was calculated as nmol/l0min/g wet weight of tissue. Values were corrected for “uptake” at 0°C which accounted for less than 10% of the radioactivity taken up in samples incubated at 37°C. Release experiments
The P,-fractions were resuspended in the uptake medium, preincubated for 5 min at 37°C and incubated for another 15 min at 37°C with 5 FM ‘H-taurine 2.7pM 14C-GABA or 0.05pM ‘H-DA, respectiveiy. Aliquots equivalent to 2&4Omg wet weight of tissue were carefully immobilized on Whatman GF/B filters by suction and washed with 30ml of medium at 37°C. The filters were transferred to a superfusion apparatus (Raiteri, Angelini and Levi, 1974) and superfused at a flow of 0.45 ml/min. After a superfusion period of 10 min to stablilize spontaneous efflux, the experiment was started at zero time and 2 min fractions of the superfusates were collected directly into liquid scintillation counting vials. After 13 min, the superfusion medium was replaced by a medium in which a part of the Na+ had been replaced isoosmotically by K+ to give a final concentration of 56mM K+. Radioactivity was determined by liquid scintillation counting and corrected for quenching by internal standardization. Release of radioactivity in 2 min fractions was expressed as percentage of the total radioactivity present on the filter at the beginning of the superfusion (total radioactivity = radioactivity remaining on filters at the end of the superfusion + radioactivity in superfusates). Stimulation-induced release during 20 min after the change to the 56mM
K+-medium was calculated by subtraction of the estimated basal release and expressed as a percentage of radioactivity present on the filters at the onset of stimulation. Assay of taurine ana’ GABA
For the determination of taurine levels striata were homogenized in 10 vol (w/v) of 10% trichloroacetic acid, centrifuged at 10,000 g for 30 min and the supernatant fluid was analyzed in a Beckman automatic amino acid analyzer (Perry, Stedman and Hansen, 1968). Concentrations of GABA were determined in aliquots of the sucrose homogenates using gas chromatography with mass fragmentographic detection as described previously (S&mid and Karobath, 1977). Materials Kainic acid, Lot 105C-0064, was obtained from Sigma Chemical Co.; [2-3H(N)]-taurine (‘Htaurine, 18 Ci/mmol) was purchased from NEN Chemicals, Dreieich; 4-amino-n-[U-“C]-butyric acid (14C-GABA, 224 mCi/mmol) and 3,4-Dihydroxy[ring-G-‘Hlphenylethylamine hydrochloride (‘H-DA, 4.9 Ci/mmol) was from the Radiochemical Center, Amersham. RESULTS Eflects of kainic acid lesions on endogenous taurine level and high ajinity uptake of 3H-taurine into crude synaptosomal (P,fiactions
Injections of kainic acid into the corpus striatum of rats significantly reduced the endogenous level of taurine and the high affinity uptake of 3H-taurine into P,-fractions by about 25% (Table 1). Parallel experiments for parameters of gabaergic and dopaminergic neurones proved the effectiveness and specificity of the
Table 1. Effect of intrastriatal injections of kainic acid on neurochemical parameters of taurine, GABA and DA
Endogenous levels : (pmol/g wet wt) Taurine GABA High affinity uptake into P,-fractions: (nmol/lO min/g wet wt) ‘H-Taurine 14C-GABA 3H-DA
Intact side
Kainic acid injected side
Percentage of intact side
P
9.4 + 0.6 3.3 + 0.3
6.8 * 0.4 0.83 + 0.12
12.2 25.1
Summary-Rats
received single unilateral stereotaxic injections of 2gg of kainic acid into the let? caudate nucfeus 10 days before sacrifices On the lesioned side, a ~~~j~~~t reduction of e~oge~o~ taurine levels and high affinity uptake of’ 3H-taurine into crude synaptosoma~ (Pa) fractions to about 75% of control vames was found. In agreement with previous reports on the nemotoxic action of uptake into P,-fractions kainic acid, a marked reduction of ~ndogeuons GABA levels and ‘%XABA was observed, while %-dapamine uptake was not sign~fi~~t~y changed. In superfusion experiments with prefabeled P2-fractions prepared from lesioned striata, K *(MmM)-induced release af “H-taurine and “*C-GABA, but not of 3H-dopamine, was reduced. The results are compatible with the hypothesis that taurine in the striatum of the rat is partially localized in interneurones.
Taurine, which is present in the brain in high concentrations, exhibits properties which indicate a possible role as neurotra~m~tter or ne~rom~ulator (for review see: Huxtable and Barbeau, 1976; Collins, 1977). It exhibits a depressant effect on neuronat activity of the cortex and brainstem of the cat (Curtis and Watkins, 1973) and it is released from the cerebral cortex on electrical stimnla?ion (Jasper and Koyama, 1969). High affinity uptake (S&mid, Sieghart and Karobath, 1975) and ~t~si~m-evoke release of taurine have been shown in rat cerebral cortex slices and synaptosomes (Kaczmarek and Davison, 1972; Sieghart and Heck], 1976; Placheta and Singer, 1977). However, these studies as well as electron microscopic autoradiography (Ehinger, 1973) or subcellular fractionation techniques with tissue cultures from glioma or neurobl~toma ceils (Sscbrier and Thompson, 1974; Sieghart and KarobatB 1976) do not ~oncl~siv~~y point to a sole neuronal localization of taurine. Recently, it has been suggested that microinjections of kainic acid inta the corpus striatum of the rat cause a selective degeneration of cholinergic and gabaergic neurones intrinsic to this region (Coyle and Schwartz, 19%; McGeer and McGeer, 1976). Therefore, if a ~onsiderabIe portion of taurine is present in striatd interneurons, Iesions with kainic acid also should lead to changes of nenro~b~rni~i parameters of &urine. In the present study, endogenous levels, high affinity uptake and release of taurine were studied using crude synaptosomal fractions derived from rat striata after intrastriatal injections of kainic acid. Key words: striatum, kainic acid, taurine, GA&A, dopamine. Abbreviations: GABA = 4-ami~o-n-b~t~jc acid, DA = dopamine.
Adult Sprague-Dawley rats of either sex were anesthetized with 50mgilcg (i.p.) ~ntobarbital. Two pg of kainic acid dissolved in 1 ,ul of buffered isotonic saline were injected stereotaxically into the head of the left caudate nucleus (coordinates: AP + 8.38; L + 2.8; DV -t 0.2; according to K&rig and Klippel, 1967), using a 0.3mm Hamilton cannnla. After 10 days the animals were decapitated and the striata of the injected and contralateral intact (control) sides were dissected on a chilled glassplate and homogenized in 10 vol (w/v) of buffered (2mM Tris-HCl, pH 7.2) icecold 0.32 M sucrose. Crude synaptosomal (Pa)fractions were prepared by conventional differentiai ~~trjfugation techniques (Whittier, 1965).
The PZ-fractions were resuspended in a medium containing in mM: NaCI, 128; KCI, 5; MgCI,, 1.2; CaCla, 0.5; d-glucose, 10; sucrose, 10; Tris-HCI, 15 (pH 7.4). Incubations with “C-GABA were performed in the presence of lO$vf aminooxyacetic acid to inhibit GABA catabolism, In the exper~ents with 3H-DA, ascorbic acid fl.14mM) and a rnono~i~ oxidase inhibitor, nialamide, (12.5pM) were included in the uptake medium. For uptake experiments aliquots of the resuspended P,-fractions, equivalent to approximately 3-6mg of original tissue wet weight, were preincubated for 5 min at 37°C in a shaking water bath under air. Then, the labeled compounds were added in 20 ,~l of medium to give a final concerttration of SpM 3H-taurine, 2.7&f ‘%X%ABA, or 0.05 yM “H-DA, respectively, (gnat volume 1.0ml) and the ~n~ubatjons were continued for another 10 min. ~~~nbations were stopped by immediate filtra-
399
400
P. PLACHETA,E.
SINGER,
G. SCH~~NJJECK, K. HECKLand M. KAROBATH
tion through cellulose nitrate filters (Schleicher and Schiill, 0.45pm pore size). The filters were rinsed 3 times with 4ml of medium at room temperature and radioactivity was determined by liquid scintillation counting. Uptake was calculated as nmol/l0min/g wet weight of tissue. Values were corrected for “uptake” at 0°C which accounted for less than 10% of the radioactivity taken up in samples incubated at 37°C. Release experiments
The P,-fractions were resuspended in the uptake medium, preincubated for 5 min at 37°C and incubated for another 15 min at 37°C with 5 FM ‘H-taurine 2.7pM 14C-GABA or 0.05pM ‘H-DA, respectiveiy. Aliquots equivalent to 2&4Omg wet weight of tissue were carefully immobilized on Whatman GF/B filters by suction and washed with 30ml of medium at 37°C. The filters were transferred to a superfusion apparatus (Raiteri, Angelini and Levi, 1974) and superfused at a flow of 0.45 ml/min. After a superfusion period of 10 min to stablilize spontaneous efflux, the experiment was started at zero time and 2 min fractions of the superfusates were collected directly into liquid scintillation counting vials. After 13 min, the superfusion medium was replaced by a medium in which a part of the Na+ had been replaced isoosmotically by K+ to give a final concentration of 56mM K+. Radioactivity was determined by liquid scintillation counting and corrected for quenching by internal standardization. Release of radioactivity in 2 min fractions was expressed as percentage of the total radioactivity present on the filter at the beginning of the superfusion (total radioactivity = radioactivity remaining on filters at the end of the superfusion + radioactivity in superfusates). Stimulation-induced release during 20 min after the change to the 56mM
K+-medium was calculated by subtraction of the estimated basal release and expressed as a percentage of radioactivity present on the filters at the onset of stimulation. Assay of taurine ana’ GABA
For the determination of taurine levels striata were homogenized in 10 vol (w/v) of 10% trichloroacetic acid, centrifuged at 10,000 g for 30 min and the supernatant fluid was analyzed in a Beckman automatic amino acid analyzer (Perry, Stedman and Hansen, 1968). Concentrations of GABA were determined in aliquots of the sucrose homogenates using gas chromatography with mass fragmentographic detection as described previously (S&mid and Karobath, 1977). Materials Kainic acid, Lot 105C-0064, was obtained from Sigma Chemical Co.; [2-3H(N)]-taurine (‘Htaurine, 18 Ci/mmol) was purchased from NEN Chemicals, Dreieich; 4-amino-n-[U-“C]-butyric acid (14C-GABA, 224 mCi/mmol) and 3,4-Dihydroxy[ring-G-‘Hlphenylethylamine hydrochloride (‘H-DA, 4.9 Ci/mmol) was from the Radiochemical Center, Amersham. RESULTS Eflects of kainic acid lesions on endogenous taurine level and high ajinity uptake of 3H-taurine into crude synaptosomal (P,fiactions
Injections of kainic acid into the corpus striatum of rats significantly reduced the endogenous level of taurine and the high affinity uptake of 3H-taurine into P,-fractions by about 25% (Table 1). Parallel experiments for parameters of gabaergic and dopaminergic neurones proved the effectiveness and specificity of the
Table 1. Effect of intrastriatal injections of kainic acid on neurochemical parameters of taurine, GABA and DA
Endogenous levels : (pmol/g wet wt) Taurine GABA High affinity uptake into P,-fractions: (nmol/lO min/g wet wt) ‘H-Taurine 14C-GABA 3H-DA
Intact side
Kainic acid injected side
Percentage of intact side
P
9.4 + 0.6 3.3 + 0.3
6.8 * 0.4 0.83 + 0.12
12.2 25.1
