GABA release and uptake measured in crude synaptosomes from Genetic Absence Epilepsy Rats from Strasbourg (GAERS)

Neurochemistry International 34 (1999) 415±425

GABA release and uptake measured in crude synaptosomes from Genetic Absence Epilepsy Rats from Strasbourg (GAERS) Rebecca J. Sutch*, Clare C. Davies, Norman G. Bowery Department of Pharmacology, Division of Neuroscience, Medical School, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK Received 22 December 1998; accepted 4 March 1999

Abstract GABA release and uptake were examined in Genetic Absence Epilepsy Rats from Strasbourg and in non-epileptic control animals, using crude synaptosomes prepared from the cerebral cortex and thalamus. Uptake of [3H]GABA over time was reduced in thalamic synaptosomes from epileptic rats, compared to controls. The anity of the uptake process in thalamic synaptosomes was lower in epileptic animals. NNC-711, a ligand for the GAT-1 uptake protein, reduced synaptosomal uptake by more than 95%; b-alanine, an inhibitor selective for the uptake proteins GAT-2 and -3, did not signi®cantly reduce synaptosomal uptake. Autoradiography studies using [3H]tiagabine, a ligand selective for GAT-1, revealed no di€erences between the strains in either anity or levels of binding. Ethanolamine O-sulphate (100 mM), a selective inhibitor of GABAtransaminase, did not a€ect uptake levels. Aminooxyacetic acid (10±100 mM), an inhibitor of GABA-transaminase and, to a lesser extent, glutamate decarboxylase, caused an increase in measured uptake in both thalamic and cortical synaptosomes, in both strains. We found no di€erence in in vitro basal or KCl-stimulated endogenous GABA release between epileptic and control rats. These results indicate that GABA uptake in the thalamus of Genetic Absence Epilepsy Rats from Strasbourg was reduced, compared to control animals. The lower uptake anity in the epileptic animals probably contributed to the reduction in uptake over time. Uptake appeared to be mediated primarily by the `neuronal' transporter GAT-1. Autoradiography studies revealed no di€erences in the number or anity of this uptake protein. It is therefore possible that altered functional modulation of GAT-1 caused the decrease in uptake shown in the epileptic animals. Inhibition of GABA-transaminase activity had no e€ect on measured GABA uptake, whereas a reduction in glutamate decarboxylase activity may have a€ected measured uptake levels. # 1999 Elsevier Science Ltd. All rights reserved.

1. Introduction Absence (petit mal) epilepsy is primarily a childhood disease, occurring most commonly in children between 4 and 11 years of age. Seizures consist of loss of consciousness characterised by a ®xed stare, immobility and lack of responsiveness to visual and verbal stimuli. Seizures begin and end abruptly, with no aura, and are common during the transition between sleep and wakefulness (Gomez and Westmoreland, 1987; Holmes et al., 1987; Drinkenburg et al., 1991; Loiseau, 1992). * Corresponding author. Tel.: +44 121-414-4519; fax: +44 121414-4509. E-mail address: [email protected] (R.J. Sutch)

They can occur as frequently as several hundred per day and are thought to be detrimental to a child's health and development (Wirrell et al., 1996, 1997). Several animal models of this disease exist, both pharmacological and genetic. Studies using these, and human subjects, indicate that absence seizures occur as a result of the generation of abnormal rhythmic oscillations between the thalamus and the cerebral cortex. These result in the appearance of characteristic Spikeand-Wave Discharges (SWD) on the EEG during an absence seizure (Williams, 1953; Avanzini et al., 1996; Seidenbecher, 1998). The oscillations may be a pathological transformation of the normal spindle oscillations that occur during sleep in order to reduce sensory input to the cortex (Steriade et al., 1993; JuhaÂsz et al., 1994; Tsoukatos et al., 1997).

0197-0186/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 1 9 7 - 0 1 8 6 ( 9 9 ) 0 0 0 4 6 - 7

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The GABA system is heavily implicated in the generation of SWD (Liu et al., 1991). In the in vitro thalamus, blockade of GABAA receptors can transform sleep spindles into rhythmic sequences of excitatory and inhibitory post-synaptic potentials similar to those seen during absence seizures (von Krosigk et al., 1993). Local and systemic applications of GABA-mimetic drugs exacerbate or induce SWD (Vergnes et al., 1984; Coenen et al., 1995) and several abnormalities relating to the GABA system have been found in genetic models of absence epilepsy (Lin et al., 1995; Luhmann et al., 1995; Richards et al., 1995; Tehrani and Barnes, 1995). The synaptic action of GABA is terminated mainly by reuptake into presynaptic terminals and glia. Four GABA transporter proteins have been cloned (GAT-1 to -4) (Guastella et al., 1990; Borden et al., 1996; Yamauchi et al., 1992). Immunocytochemical studies have shown that the rat CNS expresses mostly GAT-1, with some GAT-3 expression (Ikegaki et al., 1994; Nishimura et al., 1997). GABA is synthesised by glutamate decarboxylase (GAD) (1 glutamate 1-carboxy-lyase, EC 4.1.1.15). This enzyme is associated with synaptic terminals (Neal and Iversen, 1969) and requires pyridoxal phosphate as a coenzyme (Tapia and Covarrubias, 1978). The main GABA metabolising enzyme in the CNS is GABA-transaminase (GABA-T) (4-aminobutyrate 2oxoglutarate aminotransferase, EC 2.6.1.19). This enzyme also requires pyridoxal phosphate and is thought to be associated with mitochondria. However, synaptosomal mitochondria are believed to have little GABA-T activity (Cooper et al., 1991). Aminooxyacetic acid (AOAA) binds to pyridoxal phosphate and prevents its association with enzymes. It thus inhibits any enzyme which requires this cofactor, although it shows some selectivity for GABA-T over GAD and is commonly used to inhibit GABA-T activity. Ethanolamine O-sulphate (EOS) is a highly selective inhibitor of GABA-T, acting by irreversibly binding to the enzyme (Fowler and John, 1972). Genetic Absence Epilepsy Rats from Strasbourg (GAERS) are an inbred strain of Wistar rats which exhibit spontaneous absence seizures (Vergnes et al., 1982): 100% of animals aged 3 months show SWD. A non-epileptic control strain of rats was outbred from the same original Wistar population, and none of these show SWD (Marescaux et al., 1992). Richards et al. (1995) measured extracellular amino acid levels by in vivo microdialysis and showed that GAERS have higher levels of GABA in the thalamus than control animals. In order to examine the possible origins of this increased extracellular GABA in GAERS, we have measured GABA release and uptake in crude synaptosomes prepared from the thalamus and cortex of GAERS and controls. We have investigated the poss-

ible uptake proteins mediating the measured uptake and examined the e€ects that inhibiting the synthesis and metabolism of GABA have on uptake measurement. Finally, we have used autoradiography to quantify the CNS distribution of the uptake protein GAT-1 in GAERS and control rats. 2. Experimental procedures 2.1. Materials All reagents were of analytical grade or higher. They were all obtained from Sigma, with the following exceptions: [3H]GABA (speci®c activity 98 Ci/mmol) was from DuPont NEN; protein assay reagents were from Pierce; NNC-711 (1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid) and [3H]tiagabine ((R)-N-[4,4-bis(3-methyl-2-thienyl)but-3-en-1-yl]nipecotic acid) (speci®c activity 85 Ci/ mmol) (Braestrup et al., 1990; Suzdak et al., 1992) were generous gifts from Novo-Nordisk. 2.2. Animals Control rats and GAERS were obtained from Bantin and Kingman, Loughborough. They were housed for 24±72 h on site before being used in experiments. 2.3. Preparation of crude synaptosomes Thirteen-week-old female GAERS and control rats were killed by decapitation, after stunning. The brains were rapidly removed and the cortices and thalami dissected out on damp ice-cold ®lter paper. Brain tissues were homogenised in 0.32 M sucrose (cortices in 20 ml and thalami in 10 ml) using ten up-and-down strokes of a te¯on-glass homogeniser. Crude synaptosomes were prepared according to Hornsby et al. (1992), by a series of centrifugations. The crude synaptosomal pellets were resuspended in ice-cold arti®cial cerebrospinal ¯uid (aCSF) (containing, in mM: NaCl 126.6; NaHCO3 27.4; KCl 2.4; KH2PO4 0.49; CaCl2 1.2; MgCl2.6H2O 0.83; Na2HPO4 0.49; D-glucose 7.1; gassed with 95% O2/5% CO2; pH 7.2±7.4). Protein content of synaptosomal samples was assessed using the method of Bradford (1976). 2.4. Measurement of [3H]GABA uptake in synaptosomes Synaptosomes (0.05±0.1 mg/ml protein, ®nal vol. 1 ml/tube) were incubated at 378C. Uptake was initiated by the addition of a mixture of cold and tritiated GABA ([3H]GABA, speci®c activity 98 Ci/

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417

Fig. 1. Time courses of [3H]GABA uptake in (A) cortical and (B) thalamic synaptosomes. Synaptosomes were incubated at 378C in the presence of 25 nM GABA (10% of which was tritiated) for 0±20 min before uptake was terminated by ®ltration. Non-carrier-mediated uptake was measured in the presence of 1 mM nipecotic acid. Values are given as mean2SEM (n=5).  signi®cantly di€erent from control, P < 0.01, twoway analysis of variance on time courses.

mmol). Uptake was assayed in duplicate tubes and controls and GAERS were included in the same experiments. For time course studies, the ®nal concentration of GABA was 25 nM, 10% of which was tritiated. Initiation of uptake was staggered to allow termination at the same instant. For saturation analysis, uptake was measured at 5 min for cortex and 10 min for thalamus. The amount of [3H]GABA was kept con-

stant and the di€erent GABA concentrations (0.01± 30 mM) obtained by adding increasing concentrations of unlabelled GABA. Nipecotic acid, NNC-711, b-alanine, EOS and AOAA, where included, were preincubated with the synaptosomes 15 min before the initiation of uptake. Uptake was measured at 5 min (cortex) and 10 min (thalamus) when inhibitor e€ects were being examined. Uptake was terminated by vacuum ®ltration through

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Whatman GF/B ®lters, and the GABA content of the ®lters was assayed by liquid scintillation counting, taking dilution factors into account. Non-carrier-mediated uptake was measured in paired tubes containing 1 mM nipecotic acid, and was subtracted from total uptake to give speci®c uptake. Typically, more than 99% of cortical and 98% of thalamic uptake was prevented by nipecotic acid. All values are given as speci®c uptake. Uptake is expressed as pmol/mg protein. 2.5. Autoradiography using [3H]tiagabine Thirteen-week-old female GAERS and control animals were killed by stunning and decapitation. The brains were removed and frozen in isopentane (ÿ408C) and stored at ÿ808C until required; 12 mm horizontal sections of GAERS and control brains were thawmounted on to SuperfrostPlus slides. Autoradiography was performed according to the method of Suzdak et al. (1994), but with a 5 min wash in ice-cold Triscitrate bu€er following radioligand incubation. Slides were apposed to Hyper®lm (Amersham) for 7 days, with a microscale radiographic standard, and the resulting images analysed using micro-computerassisted densitometry. 2.6. Measurement of endogenous GABA release from perfused synaptosomes Cortical synaptosomes were resuspended in 1 ml aCSF; thalamic synaptosomes were resuspended in 0.5 ml aCSF and 100 ml aliquots (containing 00.3± 0.5 mg synaptosomal protein) were layered on to damp Whatman GF/F ®lters in separate wells of a perfusion chamber; aCSF at 208C was perfused over the synaptosomes at 0.4 ml/min. After 30 min pre-perfusion, six 2-min fractions were collected for each well. During the fourth fraction, aCSF containing 30 mM KCl (osmotically adjusted by appropriately decreasing the NaCl content) was applied for 90 s in order to stimulate release. Experiments were carried out in duplicate, with controls and GAERS in the same experiment. The endogenous GABA content of the superfusates was determined by high-performance liquid chromatography with ¯uorimetric detection, following amino acid derivitisation with O-phthalaldehyde. Release is expressed as pmol/mg protein. 2.7. Data analysis Iterative curve-®tting was done using the computer package GraphPad Prism, ®tting to the following equations, assuming one uptake/binding site: . uptake=(Vmax  time)/(Km+time)

Fig. 2. The e€ect of NNC-711 on measured uptake (A) cortical and (B) thalamic synaptosomes. Synaptosomes were preincubated for 15 min in the presence (q) or absence (Q) of NNC-711 (100 mM). Uptake of 25 nM GABA was then measured for 5 min (cortex) or 10 min (thalamus). Speci®c uptake values are given as mean2SEM (n=5).  signi®cantly di€erent from uptake without inhibitor, P < 0.001, Student's t-test.

. binding=(Bmax  concentration)/ (Kd+concentration) The same equation was used to ®t the uptake time course data. Statistical tests used were unpaired two-tailed Student's t-test, and two-factor analysis of variance: factor one was strain (control/GAERS) and factor two was time. P values less than 0.05 were considered statistically signi®cant.

3. Results 3.1. Time course of [3H]GABA uptake in crude synaptosomes Synaptosomal accumulation of [3H]GABA/GABA was time-dependent and reached a maximum level at 20 min. The rate and level of uptake was tissue-

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Table 1 Kinetic analysis of [3H]GABA uptake in synaptosomesa

Km Vmax

Cortex Control

GAERS

Thalamus Control

GAERS

4.720.8 23042169

3.620.2 19882160

2.920.5 504257

3.920.1b 573251

a

Synaptosomes were incubated with 0.01±30 mM GABA (2.5 nM [3H]GABA isotopically diluted with appropriate concentrations of GABA) for 5 min (cortex) or 10 min (thalamus). Resulting concentration-uptake curves were then analysed as described in the text to give Km and Vmax values for each experiment. The values given above are mean2SEM of ®ve separate experiments. Km values are in mM and Vmax values are in pmol/mg protein/min. b Signi®cantly di€erent from control thalamic Km, P < 0.05, Student's t-test.

speci®c, with cortical synaptosomes acquiring higher GABA levels more rapidly than thalamic nerve terminals. The time course for cortical uptake showed no di€erences between control rats and GAERS (calculated maximum uptake levels 9327 vs 8228 pmol/mg protein; time to reach 50% of uptake maximum 4.52 0.6 vs 3.920.4 min, in controls and GAERS respectively, n=5) (Fig. 1A). Thalamic uptake was signi®cantly less in GAERS compared to controls (P < 0.01, two factor analysis of variance, signi®cant e€ect of strain; n=5) (Fig. 1B). Examination of the time courses revealed that it was the amount of GABA being accumulated rather than the time taken, causing the di€erence in thalamic uptake in GAERS (maximum uptake level 74 2 15 vs 5225 pmol/mg protein; time to reach 50% of uptake maximum 6.8 2 1.0 vs 5.3 2 0.7 min, in controls and GAERS respectively, n=4±5). 3.2. Synaptosomal uptake was inhibited by NNC-711 but not by b-alanine The GAT-1 selective inhibitor NNC-711 virtually abolished carrier-mediated [3H]GABA uptake (Fig. 2). A small level of uptake remained in the presence of 100 mM NNC-711: 1.920.1 and 2.020.3% of control cortical uptake levels; 5.120.8 and 4.920.8% of control thalamic uptake levels, control rats and GAERS respectively (n=6). Nevertheless, 95% of thalamic and 98% of cortical uptake was blocked by NNC-711, with the same level of inhibition occurring in GAERS and controls. Dose-response studies with NNC-711 revealed no di€erences in IC50 values between the rat strains (0.12 20.02 vs 0.1020.02 mM in cortex and 0.11 vs 0.092 0.01 mM in thalamus, control rats vs GAERS, n=3 except for control thalamic uptake where n=2); 10 mM NNC-711 produced almost identical levels of inhibition as 100 mM (data not shown). b-Alanine, at concentrations up to 100 mM, had no

signi®cant e€ect on GABA uptake. The percentage inhibition values were: 923 and 723% in cortex; 921 and 1623% in thalamus; control and GAERS values respectively, in 7±8 separate experiments. 3.3. Kinetic analysis of GABA uptake in crude synaptosomes Incubation of cortical and thalamic synaptosomes with increasing concentrations of GABA (0.1±30 mM) resulted in hyperbolic concentration-uptake curves. Analysis of these produced the kinetic parameters Km and Vmax (Table 1). Vmax values were higher in cortical compared to thalamic synaptosomes. However, there was no di€erence between GAERS and controls in either thalamic or cortical Vmax values. Km values in the cortex

Table 2 (A) Kd and (B) Bmax values for [3H]tiagabine binding to brain slicesa

A Cortex Ventral thalamus Medial thalamus Lateral geniculate nucleus Hippocampus B Cortex Ventral thalamus Medial thalamus Lateral geniculate nucleus Hippocampus

Control

GAERS

2823 4926 3027 3628 2524

2523 3526 3025 2726 2622

1245270 719269 9572122 10452139 11782124

14232100 7512111 1021272 9692115 13612103

a Autoradiography using [3H]tiagabine concentrations of 3±300 nM was carried out as described in the text. Iterative curve ®tting yielded Bmax and Kd values for each animal. Values are given as mean2SEM, n=5. Kd values are in mM, Bmax values are in fmol/mg protein. No di€erences were detected between controls and GAERS.

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Fig. 3. Time courses of [3H]GABA uptake in (A) cortical and (B) thalamic synaptosomes in the presence of EOS. Synaptosomes (R controls and q GAERS) were preincubated at 378C with ethanolamine O-sulphate (100 mM) for 15 min before being incubated in the presence of 25 nM GABA (10% of which was tritiated) for 0±20 min. Non-carrier-mediated uptake was measured in the presence of 1 mM nipecotic acid. Values are given as mean 2 SEM (n=3). No di€erences were detectable between uptake values in the presence of EOS compared to control time courses.

showed no di€erence between GAERS and controls. GAERS thalamic synaptosomes showed a higher Km than control thalamic synaptosomes (P < 0.05, Student's t-test, n=5). 3.4. Autoradiographical analysis of [3H]tiagabine binding Autoradiography saturation binding was performed with 3±300 nM [3H]tiagabine, and produced Kd and

Fig. 4. The e€ect of AOAA on measured uptake in (A) cortical and (B) thalamic synaptosomes. Synaptosomes were preincubated for 15 min in the absence (Q) or presence of 10 mM (q) or 100 mM (+) AOAA. Uptake of 25 nM GABA was then measured for 5 min (cortex) or 10 min (thalamus). Speci®c uptake values are given as mean2 SEM (n=4±6).  signi®cantly di€erent from uptake without AOAA, P < 0.05, Student's t-test.

Bmax values as described in Table 2A and B respectively. No di€erences in these parameters were detectable between control and GAERS. 3.5. The e€ect of ethanolamine O-sulphate on measured GABA uptake EOS (100 mM) produced no signi®cant changes in the amount of GABA taken up by either thalamic or cortical preparations at any time point (Fig. 3). However, the calculated maximum uptake level was signi®cantly higher in control cortical synaptosomes, compared to that calculated for experiments in the absence of EOS (162233 vs 9327 pmol/mg protein/ min, P < 0.05, Student's t-test, n=3 for EOS exper-

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421

Table 3 Endogenous GABA release from cortical and thalamic synaptosomesa

Basal Stimulated

Cortex Control

GAERS

Thalamus Control

GAERS

7828.7 214223

6329.3 214238

138214 330238

119215 288247

a Synaptosomes were perfused as described in the text: 2-min fractions were collected after 30 min perfusion. Basal release was calculated for each experiment as the mean GABA content of the ®rst three fractions. Stimulated release was induced by 30 mM KCl during the fourth fraction. Values are pmol GABA/mg protein/2 min, mean2SEM (n=4 for cortex and 7 for thalamus). No di€erences in release were detected between controls and GAERS.

AOAA signi®cantly increased measured uptake in both areas and both strains (Fig. 4). The magnitude of the increase was similar in all preparations: 12324% vs 13422% in cortex; 13425% vs 13925% in thalamus, control vs GAERS (n=6). 3.7. Uptake measured at 30 min in the presence and absence of AOAA

Fig. 5. The e€ect of AOAA on measured uptake in (A) cortical and (B) thalamic synaptosomes. Synaptosomes were preincubated for 15 min in the absence (Q) or presence of 10 mM (q) or 100 mM (+) AOAA. Uptake of 25 nM GABA was then measured for 30 min in cortex and thalamus. Speci®c uptake values are given as mean 2 SEM (n=4).  signi®cantly di€erent from corresponding uptake in control rats, P < 0.05;  signi®cantly di€erent from corresponding uptake in control rats, P < 0.005; % signi®cantly di€erent from uptake without AOAA, P < 0.001, Student's t-test.

iments). Cortical synaptosomes from GAERS and thalamic synaptosomes from both strains showed no signi®cant increase in their uptake maxima, although there appeared to be a trend towards an increase in the calculated maximum values. The time course of uptake was altered in both cortical and thalamic preparations: the times taken to accumulate 50% of uptake maxima were 13.5 2 2.1 and 10.122.4 min in cortex; 15.621.4 and 13.720.4 min in thalamus, controls and GAERS respectively (n=3). These values are signi®cantly higher (P < 0.02, Student's t-test), for both regions and both strains, than those measured in the absence of EOS. 3.6. The e€ects of AOAA on measured GABA uptake at 5 or 10 min 10 mM AOAA had no signi®cant e€ects on measured GABA uptake (n=4). However, 100 mM

After reaching a maximum at 20 min, measured GABA uptake levels began to decline with time in both thalamic and cortical synaptosome preparations. Measurement of uptake without AOAA at the single time point of 30 min incubation revealed signi®cant di€erences in GAERS uptake compared to control animals in both thalamic and cortical synaptosomes (Fig. 5). In the presence of 10 mM AOAA, this di€erence at 30 min was exaggerated without any signi®cant change in uptake levels (in control animals, P = 0.08 for cortical and P = 0.07 for thalamic uptake levels compared to no AOAA present, n=4) (Fig. 5). In the presence of 100 mM AOAA, uptake at 30 min was signi®cantly increased in all preparations (Fig. 5). The di€erence in cortical uptake between controls and GAERS was abolished when 100mM AOAA was present. The magnitude of the di€erence between GAERS and control thalamic uptake was maintained, but failed to achieve statistical signi®cance (P = 0.06, n=5). 3.8. Release of endogenous GABA from cortical and thalamic synaptosomes Levels of GABA release were examined in perfused crude synaptosomes prepared from thalami and cortices of control rats and GAERS. Release levels observed in cortical synaptosomes were lower than those seen in thalamic synaptosomes: 30 mM KCl applied for 90 s induced release levels approximately

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240% of basal in thalamus, and 280±330% of basal in cortex. No di€erences between controls and GAERS could be detected in levels of basal or KCl-stimulated release (Table 3). 4. Discussion 4.1. Measurement of uptake over time in synaptosomes Isolated synaptosomes accumulate [3H]GABA in a concentration- and time-dependent manner (Neal and Iversen, 1969). Iversen and Kelly (1975) proposed that the mechanism involved is one of high anity uptake involving nerve terminals which normally contain and synthesise GABA. Others have suggested that a homoexchange mechanism is occurring (Levi et al., 1978). In homoexchange, for each molecule of [3H]GABA taken up, one molecule of endogenous GABA is displaced into the extracellular medium. For both homoexchange and high anity uptake, metabolism of GABA is a potential problem. Tritiated metabolites are not retained within synaptosomes so that any [3H]GABA taken up and then metabolised is e€ectively `lost' and is not measured (Iversen and Neal, 1968; Neal and Starr, 1973). Before interpreting the di€erences in uptake between GAERS and control rats, the in¯uences of GABA-T activity, GABA release, and GAD activity on measured uptake were considered. 4.2. Release of endogenous GABA from crude synaptosomes GABA release was measured using a perfusion system, to eliminate reuptake di€erences (Raiteri et al., 1974). Depolarisation-evoked release was elicited by 30 mM KCl in the perfusion medium. Neither basal nor stimulated release levels showed any di€erence between GAERS and control rats, in either cortex or thalamus. It is therefore unlikely that endogenous GABA released during uptake measurement a€ected [3H]GABA uptake to di€erent extents in the di€erent strains. 4.3. E€ect of GABA-T activity on measured uptake In cortical slices, levels of measured uptake decrease after 020 min incubation, probably as a result of [3H]GABA breakdown by GABA-T (Iversen and Neal, 1968). In our hands neither EOS, at a concentration reported to inhibit up to 80% of GABA-T activity in rat retinae (Starr, 1975), nor AOAA (10 mM) signi®cantly altered the uptake of [3H]GABA into rat cortical or thalamic synaptosomes. Neither compound, at the concentrations used, interfered directly with the

uptake process (LoÈscher, 1980). Other work with rat retinae has shown that the e€ects of GABA-T are not apparent at less than 30 min uptake incubation time (Neal and Starr, 1973), and our data from crude synaptosomes were in agreement with this. There was some indication in synaptosomes from control rats that GABA-T was beginning to exert its e€ects at 30 min. The apparent shift in all of the uptake time courses in the presence of EOS was probably due to the higher values calculated for the uptake maxima. However, blockade of GABA-T activity appeared to have no e€ect on uptake levels measured at 0±20 min. 4.4. E€ect of 100 mM AOAA on measured uptake AOAA concentrations as low as 1 mM have been reported to cause measurable changes in [3H]GABA uptake due to inhibition of GABA-T activity; 100 mM AOAA has been reported to also inhibit GAD activity by 75±80% (Neal and Starr, 1973; Starr, 1975). This additional inhibition may explain the large increase in uptake caused by 100 mM AOAA observed at both early and later time points, but why this e€ect would result in an increase in measured uptake levels is unclear. Perhaps endogenous GABA was being displaced out of the synaptosomes, as suggested by the homoexchange model, and was diluting the tritiated GABA in the medium, leading to underestimation of [3H]GABA uptake. Inhibition of GAD may have reduced the pool of endogenous GABA available to exchange in this way. It has also been suggested that GAD activity is linked directly to GABA release (Tapia and Covarrubias, 1978), and inhibition of GAD may therefore reduce GABA leakage into the medium and decrease the underestimation of uptake levels. AOAA also a€ects other enzymes dependent on pyridoxal phosphate as a coenzyme, e.g. other aminotransferases (Starr and Tanner, 1975). However, it seems unlikely that inhibition of these enzymes would have such a rapid and dramatic e€ect on GABA uptake levels, as their action would be indirect. It seems more likely that the increase in uptake caused by 100 mM AOAA was linked to its additional inhibition of GAD, and that GAD activity was somehow reducing measured levels of uptake. 4.5. The transporter proteins mediating uptake in synaptosomes NNC-711 is highly selective for GAT-1 (Borden et al., 1994). It has been suggested that 10 mM NNC-711 is sucient to completely inhibit GAT-1 mediated uptake into transfected cells, with less than 10% inhibition of GAT-3 (Clark et al., 1992). In our uptake assay, virtually all GABA uptake was blocked at this

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concentration. Reported IC50 values range from 0.05± 0.4 mM (Suzdak et al., 1992; Borden et al., 1994). Our ®nding of 00.1 mM IC50 is in agreement with these values. b-Alanine is a weak inhibitor of GAT-1 (IC50 3 mM), whereas it will inhibit the rat uptake proteins GAT-2 and GAT-3 by 60±90% at a concentration of 100 mM (Liu et al., 1993; Clark et al., 1992; Borden et al., 1996, 1994). At this concentration, b-alanine caused no inhibition of [3H]GABA uptake into synaptosomes. Although GAT-3 expression has been described in the thalamus (Ikegaki et al., 1994; Nishimura et al., 1997; Durkin et al., 1995), its neuronal/glial distribution there has not been examined. It has been proposed that neurones express only GAT-1, whereas GAT-3 is expressed by astrocytes (Itouji et al., 1996; Minelli et al., 1996; Nishimura et al., 1997). As synaptosomes are isolated nerve terminals, only neuronal uptake proteins would be expected to mediate synaptosomal uptake. This may explain why over 95% of the measured uptake was apparently GAT-1mediated. 4.6. Kinetic analysis of uptake Uptake Vmax values represent the number of transport molecules present in the synaptosomal preparations. Cortical Vmax values were higher than thalamic values, probably re¯ecting the higher density of GAT-1 sites in the cerebral cortex compared to thalamus (cf autoradiography data, below, and Suzdak et al., 1994). Clark et al. (1992) reported a Km value of 2.3 mM for the cloned GAT-3 protein. Guastella et al. (1990) reported Km values for the cloned GAT-1 transporter of 3±11 mM. Km values measured here were all 03 mM, indicating a high anity uptake system was responsible for [3H]GABA accumulation. This Km value does not allow distinction between GAT-1 or GAT-3 but the results with NNC-711 are consistent with the involvement of GAT-1. 4.7. Autoradiography using [3H]tiagabine Tiagabine binds to GAT-1 with high selectivity (Borden et al., 1994). [3H]tiagabine has been used previously to quantify the distribution of GABA uptake sites in the rat CNS (Suzdak et al., 1994). This group reported high levels of binding in the cerebral cortex and hippocampus, slightly lower levels in the thalamus, and a Kd value of 060 nM. The results reported here are in general agreement with this distribution and anity and no di€erences in Kd and Bmax were apparent between control rats and GAERS.

423

4.8. Comparison of cortical uptake in GAERS and control rats Although a di€erence in GABA-T activity may explain the lower level of measured uptake in GAERS cortex at 30 min, it does not explain why 10 mM AOAA did not abolish the di€erence, nor does it explain why the divergence of the cortical time courses was still apparent in the presence of EOS. The di€erence may best be explained as an increase in GAD activity in GAERS' cortex, leading to a progressively greater underestimation of uptake in cortical synaptosomes from these animals. 4.9. Comparison of thalamic uptake in GAERS and control rats GAERS thalamic uptake was consistently 075% of control levels at all time points, although only uptake at 30 min was signi®cantly di€erent; 100 mM AOAA increased uptake by the same extent in both strains, suggesting that GABA-T and GAD activities are similar in the thalamus of control rats and GAERS. Assessment of Vmax revealed no di€erence in maximum uptake rate between GAERS and controls, suggesting that the number of uptake sites was unaltered in GAERS. Km was signi®cantly higher in GAERS thalamic synaptosomes, compared to control rats, indicating a lower anity of the transport proteins for GABA. The reduction in anity for GABA contributed to the reduction in uptake over time observed in GAERS thalamic synaptosomes. Although autoradiography revealed no di€erences in the anity of [3H]tiagabine for GAT-1, tiagabine does not bind to the same site on the transporter molecule as GABA, nor is it a substrate for uptake (Braestrup et al., 1990) and therefore this ligand might not detect functional changes in uptake. Functional modulation of GAT-1 by protein kinase C has been reported, both as upregulation by changes to Vmax (Corey et al., 1994; Quick et al., 1997; Beckman et al., 1998) and downregulation by changes to Km (Gomeza et al., 1991; Osawa et al., 1994; Sato et al., 1995). GAT-1 also contains consensus phosphorylation sites for protein kinase A (Guastella et al., 1990). It seems possible, therefore, that thalamic GAT1 in GAERS is in a di€erent phosphorylation state and therefore shows reduced anity for GABA, compared to non-epileptic animals. References Avanzini, G., de Curtis, M., Franceschetti, S., Sancini, G., Sprea®co, R., 1996. Cortical versus thalamic mechanisms underlying spike and wave discharges in GAERS. Epilepsy Res. 26, 37±44.

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R.J. Sutch et al. / Neurochemistry International 34 (1999) 415±425

Beckman, M.L., Bernstein, E.M., Quick, M.W., 1998. Protein kinase C regulates the interaction between a GABA transporter and syntaxin 1A. J. Neurosci. 18, 6103±6112. Borden, L.A., 1996. GABA transporter heterogeneity: pharmacology and cellular localization. Neurochem. Int. 29, 335±356. Borden, L.A., Murali Dhar, T.G., Smith, K.E., Weinshank, R.L., Branchek, T.A., Gluchowski, C., 1994. Tiagabine, SK&F 89976A, CI-966, and NNC-711 are selective for the cloned GABA transporter GAT-1. Eur. J. Pharmacol. 269, 219±224. Bradford, M.M., 1976. A rapid and sensitive method for the quanti®cation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochem. 72, 248±254. Braestrup, C., Nielsen, E.B., Sonnewald, U., Knutsen, L.J., Andersen, K.E., Jansen, J.A., Frederiksen, K., Andersen, P.H., Mortensen, A., Suzdak, P.D., 1990. (R)-N-[4,4-bis(3-methyl-2thienyl)but-3-en-1-yl]nipecotic acid binds with high anity to the brain gamma-aminobutyric acid uptake carrier. J. Neurochem. 54, 639±647. Clark, J.A., Deutch, A.Y., Gallipoli, P.Z., Amara, S.G., 1992. Functional expression and CNS distribution of a beta-alaninesensitive neuronal GABA transporter. Neuron 9, 337±348. Coenen, A.M.L., Blezer, E.H.M., Vanluijtelaar, E.L.J.M., 1995. E€ects of the gaba-uptake inhibitor tiagabine on electroencephalogram, spike-wave discharges and behavior of rats. Epilepsy Res. 21, 89±94. Cooper, J.R., Bloom, F.E., Roth, R.H., 1991. Amino acid neurotransmitters. In: Cooper, J.R., Bloom, F.E., Roth, R.H. (Eds.), The Biochemical Basis of Neuropharmacology. Oxford University Press, Oxford, pp. 113±189. Corey, J.L., Davidson, N., Lester, H.A., Brecha, N., Quick, M.W., 1994. Protein kinase C modulates the activity of a cloned gamma-aminobutyric acid transporter expressed in Xenopus oocytes via regulated subcellular redistribution of the transporter. J. Biol. Chem. 269, 14,759±14,767. Drinkenburg, W.H., Coenen, A.M., Vossen, J.M., Van Luijtelaar, E.L., 1991. Spike-wave discharges and sleep-wake states in rats with absence epilepsy. Epilepsy Res. 9, 218±224. Durkin, M.M., Smith, K.E., Borden, L.A., Weinshank, R.L., Branchek, T.A., Gustafson, E.L., 1995. Localization of messenger RNAs encoding three GABA transporters in rat brain: an in situ hybridization study. Brain Res. Mol. Brain Res. 33, 7±21. Fowler, L.J., John, R.A., 1972. Active-site-directed irreversible inhibition of rat brain 4-aminobutyrate aminotransferase by ethanolamine O-sulphate in vitro and in vivo. Biochem. J. 130, 569± 573. Gomez, M.R., Westmoreland, B.F., 1987. Absence seizures. In: Luders, H., Lesser, R.P. (Eds.), EpilepsyÐElectroclinical Syndromes. Springer±Verlag, pp. 105±129. Gomeza, J., Casado, M., Gimenez, C., Aragon, C., 1991. Inhibition of high-anity gamma-aminobutyric acid uptake in primary astrocyte cultures by phorbol esters and phospholipase C. Biochem. J. 275, 435±439. Guastella, J., Nelson, N., Nelson, H., Czyzyk, L., Keynan, S., Miedel, M.C., Davidson, N., Lester, H.A., Kanner, B.I., 1990. Cloning and expression of a rat brain GABA transporter. Science 249, 1303±1306. Holmes, G.L., McKeever, M., Adamson, M., 1987. Absence seizures in children: clinical and electroencephalographic features. Ann. Neurol. 21, 268±273. Hornsby, C.D., Barnes, J.M., Barnes, N.M., Champaneria, S., Costall, B., Naylor, R., 1992. Pharmacological comparison of the rat and guinea pig cortical high-anity 5-hydroxytryptamine uptake system. Biochem. Pharmacol. 43, 1865±1868. Ikegaki, N., Saito, N., Hashima, M., Tanaka, C., 1994. Production of speci®c antibodies against GABA transporter subtypes (GAT1, GAT2, GAT3) and their application to immunocytochemistry. Brain Res. Mol. Brain Res. 26, 47±54.

Itouji, A., Sakai, N., Tanaka, C., Saito, N., 1996. Neuronal and glial localization of two GABA transporters (GAT1 and GAT3) in the rat cerebellum. Brain Res. Mol. Brain Res. 37, 309±316. Iversen, L.L., Kelly, J.S., 1975. Uptake and metabolism of gammaaminobutyric acid by neurones and glial cells. Biochem. Pharmacol. 24, 933±938. Iversen, L.L., Neal, M.J., 1968. The uptake of [3H]GABA by slices of rat cerebral cortex. J. Neurochem. 15, 1141±1149. Juhasz, G., Emri, Z., Kqkesi, K.A., Salfay, O., Crunelli, V., 1994. Blockade of thalamic GABAB receptors decreases EEG synchronization. Neurosci. Lett. 172, 155±158. Levi, G., Banay-Schwartz, M., Raiteri, M., 1978. Uptake, exchange and release of GABA in isolated nerve endings. In: Fonnum, F. (Ed.), Amino acids as chemical transmitters. Plenum Press, pp. 327±350. Lin, F.H., Wang, Y., Lin, S., Cao, Z., Hosford, D.A., 1995. GABAB receptor-mediated e€ects in synaptosomes of lethargic (lh/lh) mice. J. Neurochem. 65, 2087±2095. Liu, Q.R., Lopez-Corcuera, B., Mandiyan, S., Nelson, H., Nelson, N., 1993. Molecular characterization of four pharmacologically distinct gamma-aminobutyric acid transporters in mouse brain. J. Biol. Chem. 268, 2106±2112 (corrected: published erratum appears in J. Biol. Chem. Apr 25;268(12): 9156). Liu, Z., Vergnes, M., Depaulis, A., Marescaux, C., 1991. Evidence for a critical role of GABAergic transmission within the thalamus in the genesis and control of absence seizures in the rat. Brain Res. 545, 1±7. Loiseau, P., 1992. Human absence epilepsies. J. Neural. Transm. Suppl. 35, 1±6. LoÈscher, W., 1980. E€ect of inhibitors of GABA transaminase on the synthesis, binding, uptake and metabolism of GABA. J. Neurochem. 34 (6), 1603±1608. Luhmann, H.J., Mittmann, T., van Luijtelaar, G., Heinemann, U., 1995. Impairment of intracortical GABAergic inhibition in a rat model of absence epilepsy. Epilepsy Res. 22, 43±51. Marescaux, C., Vergnes, M., Depaulis, A., 1992. Genetic absence epilepsy in rats from StrasbourgÐa review. J. Neural. Transm. Suppl. 35, 37±69. Minelli, A., DeBiasi, S., Brecha, N.C., Zuccarello, L.V., Conti, F., 1996. GAT-3, a high-anity GABA plasma membrane transporter, is localized to astrocytic processes, and it is not con®ned to the vicinity of GABAergic synapses in the cerebral cortex. J. Neurosci. 16, 6255±6264. Neal, M.J., Iversen, L.L., 1969. Subcellular distribution of endogenous and [3H]g-aminobutyric acid in rat cerebral cortex. J. Neurochem. 16, 1245±1252. Neal, M.J., Starr, M.S., 1973. E€ect of inhibitors of g-aminobutyrate aminotransferase on the accumulation of [3H]-g-aminobutyric acid by the retina. Brit. J. Pharmacol. 47, 543±555. Nishimura, M., Sato, K., Mizuno, M., Yoshiya, I., Shimada, S., Saito, N., Tohyama, M., 1997. Di€erential expression patterns of GABA transporters (GAT1-3) in the rat olfactory bulb. Mol. Brain Res. 45, 268±274. Osawa, I., Saito, N., Koga, T., Tanaka, C., 1994. Phorbol esterinduced inhibition of gaba uptake by synaptosomes and by xenopus-oocytes expressing gaba transporter (GAT1). Neurosci. Res. 19, 287±293. Quick, M.W., Corey, J.L., Davidson, N., Lester, H.A., 1997. Second messengers, tracking-related proteins, and amino acid residues that contribute to the functional regulation of the rat brain GABA transporter GAT1. J. Neurosci. 17, 2967±2979. Raiteri, M., Angelini, F., Levi, G., 1974. A simple apparatus for studying the release of neurotransmitters from synaptosomes. Eur. J. Pharmacol. 25, 411±414. Richards, D.A., Lemos, T., Whitton, P.S., Bowery, N.G., 1995. J. Neurochem. 65, 1674±1680 (Abstract). Sato, K., Betz, H., Schloss, P., 1995. The recombinant GABA trans-

R.J. Sutch et al. / Neurochemistry International 34 (1999) 415±425 porter GAT1 is downregulated upon activation of protein kinase C. FEBS Lett. 375, 99±102. Seidenbecher, T., Staak, R., Pape, H-C., 1998. Relations between cortical and thalamic cellular activities during absence seizures in rats. Eur. J. Neurosci. 10, 1103±1112. Starr, M.S., 1975. E€ects of metabolic inhibitors on amino acid metabolism in rat retina: a comparison of amino-oxyacetic acid and ethanolamine O-sulphate. J. Neurochem. 24, 1229±1236. Starr, M.S., Tanner, T., 1975. E€ects of aminooxyacetic acid, ethanolamine O-sulphate and GABA on the contents of GABA and various amines in brain slices. J. Biochem. 25, 573±577. Steriade, M., McCormick, D.A., Sejnowski, T.J., 1993. Thalamocortical oscillations in the sleeping and aroused brain. Science 262, 679±685. Suzdak, P.D., Foged, C., Andersen, K.E., 1994. Quantitative autoradiographic characterization of the binding of [h-3]-tiagabine (nnc-05-328) to the gaba uptake carrier. Brain Res. 647, 231±241. Suzdak, P.D., Frederiksen, K., Andersen, K.E., Sc° rensen, P.O., Knutsen, L.J., Nielsen, E.B., 1992. NNC-711, a novel potent and selective gamma-aminobutyric acid uptake inhibitor: pharmacological characterization. Eur. J. Pharmacol. 224, 189±198. Tapia, R., Covarrubias, M., 1978. Glutamate decarboxylase, properties and the synaptic function of GABA. In: Fonnum, F. (Ed.), Amino acids as chemical transmitters. Plenum Press, pp. 431±438. Tehrani, M.H., Barnes Jr, E.M., 1995. Reduced function of gammaaminobutyric acidA receptors in tottering mouse brain: role of cAMP-dependent protein kinase. Epilepsy Res. 22, 13±21. Tsoukatos, J., Kiss, Z.H.T., Davis, K.D., Tasker, R.R., Dostrovsky,

425

J.O., 1997. Patterns of neuronal ®ring in the human lateral thalamus during sleep and wakefulness. Exp. Brain Res. 113, 273±282. Vergnes, M., Marescaux, C., Micheletti, G., Depaulis, A., Rumbach, L., Warter, J.M., 1984. Enhancement of spike and wave discharges by GABAmimetic drugs in rats with spontaneous petitmal-like epilepsy. Neurosci. Lett. 44, 91±94. Vergnes, M., Marescaux, C., Micheletti, G., Reis, J., Depaulis, A., Rumbach, L., Warter, J.M., 1982. Spontaneous paroxysmal electroclinical patterns in rat: a model of generalized non-convulsive epilepsy. Neurosci. Lett. 33, 97±101. von Krosigk, M., Bal, T., McCormick, D.A., 1993. Cellular mechanisms of a synchronized oscillation in the thalamus. Science 261, 361±364. Williams, D., 1953. A study of thalamic and cortical rhythms in petit mal. Brain 76, 50±69. Wirrell, E.C., Cam®eld, C.S., Cam®eld, P.R., Dooley, J.M., Gordon, K.E., Smith, B., 1997. Long-term psychosocial outcome in typical absence epilepsyÐSometimes a wolf in sheep's clothing. Arch. Pediatr. Adolesc. Med. 151, 152±158. Wirrell, E.C., Cam®eld, C.S., Cam®eld, P.R., Gordon, K.E., Dooley, J.M., 1996. Long-term prognosis of typical childhood absence epilepsy: Remission or progression to juvenile myoclonic epilepsy. Neurology 47, 912±918. Yamauchi, A., Uchida, S., Kwon, H.M., Preston, A.S., Robey, R.B., Garcia-Perez, A., Burg, M.B., Handler, J.S., 1992. Cloning of an Na(+)- and Cl(ÿ)-dependent betaine transporter that is regulated by hypertonicity. J. Biol. Chem. 267, 649±652.