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Plasticity of amino acid release and uptake during kindling epileptogenesis

Zuiderwijk, M.

Publication date2000

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Citation for published version (APA):Zuiderwijk, M. (2000). Plasticity of amino acid release and uptake during kindlingepileptogenesis.

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Chapte rr 2

Biochemica ll measuremen t of electrically-stimulate dd GABA releas e in

thee CA1 regio n of hippocampa l slice s

30 0

Electrically-stimulatedElectrically-stimulated GAB A release in CA1 region

Biochemica ll measuremen t of electrically-stimulate d GABAA releas e in the CA1 regio n of hippocampa l slices *

Abstrac t t

AA biochemical method to measure local presynaptic GABA release induced by

electricall stimulation of the CA1 region of hippocampal slices was developed.

GABAA release, induced by repetitive short-lasting (3-s) tetanic stimulation of

Schaffer-collateral/commissurall fibres, was measured in 1-min fractions collected

viaa a cannula placed just above the stratum radiatum. GABA release was

increasedd upon tetanic stimulation, but this was only observed in the presence of

thee GABA uptake inhibitor SK&F 89976-A. The stimulated GABA release was

restrictedd to the CA1 region and was fully Ca2 +-dependent, indicating its

presynapticc vesicular origin. Application of the selective G A B A B autoreceptor

antagonistt CGP 52432 enhanced the tetanically-stimulated GABA release,

indicatingg active regulation of GABA release by presynaptic G A B A B

autoreceptorss during high-frequency stimulation. This study demonstrates that

regulationn of local electrically-evoked presynaptic GABA release in the CA1

regionn can be measured.

Too be submitted for publication

31 1

ChapterChapter 2

Introductio n n

GABA-mediatedd inhibition is important to maintain the balance between

excitatoryy and inhibitory synaptic transmission in the hippocampal network.

Evidencee is accumulating that changes in GABAergic transmission in the

hippocampuss mediate processes of synaptic plasticity, such as learning and

memoryy (Bliss & Collingridge, 1993; Paulsen & Moser, 1998) and epilepsy

(Bradford,, 1995; Lopes da Silva et al., 1995). As such, changes in presynaptic

GABAA release from hippocampal tissue in these processes have been investigated

neurochemica l̂̂ in several studies, both in vivo (During et al., 1992, 1995) and in

vitrovitro (Kamphuis et al., 1991b; Ghijsen et al., 1992). In these studies GABA release

wass evoked by depolarization with high K+, which makes identification of the

sourcee of released GABA and the synaptic pathways involved rather difficult .

Furthermore,, the nature of this depolarizing stimulus is very different from that

occurringg under physiological conditions. To overcome these limitations, local

presynapticc GABA release was investigated using single-cell patch-clamp

recordingg (Bekkers, 1994). Using this approach, spontaneous miniature inhibitory

postsynapticc currents were quantified. However, this method is rather indirect

andd depends on postsynaptic receptor activity and density (Nusser et al., 1998).

Therefore,, a direct measure of presynaptic transmitter release would be

preferable.. A few biochemical studies have measured release of endogenous

aminoo acids from hippocampal slices induced by local high-frequency electrical

stimulationn of Schaffer-collateral/commissural fibres (Spencer et al., 1981; Roisin

ett al., 1991; Klancnik et al., 1992). Although these studies have reported increases

inn electrically-stimulated aspartate and glutamate release, changes in GABA

releasee were not observed. Corradetti et al. (1983) measured enhanced GABA

releasee upon Schaffer-collateral fibre stimulation, but this was not measured

locallyy since superfusion fluid from the whole hippocampal slice was collected.

Moreover,, in all these studies continuous stimulation was applied, lasting

severall minutes, which is not very physiological. Repetitive short-lasting high-

frequencyy stimuli have been shown to enhance GABA release, but only in striatal

slicess (Calabresi et al., 1995).

Inn the present study, we developed a method to measure local presynaptic

GABAA release biochemically from the CA1 region of hippocampal slices upon

repe t i t i vee shor t - las t ing h igh- f requency s t imula t ion of Schaffer-

collateral/commissurall fibres. GABA release was measured with a cannula

positionedd just above the apical dendritic field (stratum radiatum) of the CA1

32 2

Electrically-stimulatedElectrically-stimulated GABA release in CA1 region

region,, where the Schaffer-collaterals project upon. Since the amount of GABA

collectedd by the cannula is the result of release and re-uptake by high-affinity

GABAA transporters present in the CA1 region (Borden, 1996), possible changes in

GABAA release locally around synapses could be masked by rapid clearance.

Therefore,, experiments were performed both in the absence and in the presence

off the GABA uptake inhibitor SK&F 89976-A (Yunger et al., 1984). The regulation

off the high-frequency-stimulated GABA release by G A B A B autoreceptors was

investigatedd using the broad G A B A B receptor antagonist saclofen (Kerr et al.,

1989)) and the more selective G A B A B autoreceptor antagonist CGP 52432 (Lanza et

al.,, 1993).

Thiss method can be useful to investigate alterations in the regulation of

presynapticc GABA release in processes of synaptic plasticity in the hippocampus,

suchh as epilepsy.

Material ss and method s

Chemicals Chemicals

SK&FF 89976-A was kindly provided by Dr. Skidmore (Smith, Klin e &

Beecham,, Welwyn, UK). Saclofen was purchased from Tocris Cookson (Bristol,

UK).. CGP 52432 was a generous gift from Dr. Froestl (Novartis Pharma, Basel,

Switzerland).. Hypersil was from Shandon (Applied Science Group, Emmen, The

Netherlands).. Al l other chemicals were of the purest grade available and were

obtainedd from Sigma (Brunschwig, Amsterdam, The Netherlands) and Merck

(Amsterdam,, The Netherlands). Al l solutions were prepared with HPLC-grade

ultra-puree water generated by a "Milli-Q " purification system (Millipore, Bedford,

MA ,, USA).

PreparationPreparation of hippocampal slices

Forr the experiments, male Wistar rats (100-180 g body weight) were used. On

thee experimental day, the animals were anaesthetized with ketamine (0.1 ml/100

gg body weight, i.p.) and decapitated. The hippocampus was dissected out and 500

fimfim thick transversal slices from the dorsal hippocampus were cut with a tissue

chopper.. The slices were allowed to equilibrate for at least 1 h at room

temperaturee in a holding chamber filled with artificial cerebrospinal fluid (a-

33 3

ChapterChapter 2

CSF),, containing (in mM) 124 NaCl, 3.5 KC1, 2 CaCl2, 2 MgS04,1.25 NaH2P04, 26

NaHC03,100 D-glucose, gassed with a mixture of 95 % O2 and 5 % CÜ2, at pH 7.4. A

singlee slice was transferred to a recording chamber (of about 1.0 ml volume),

coveredd by a nylon grid, and submerged in continuously flowing a-CSF (1.0

ml /min)) at a temperature of 31-33°C.

ElectricalElectrical stimulation

AA bipolar stimulation electrode (stainless steel wires, 60 film diameter) was

posit ionedd in the Schaffer-collateral/commissural fibre pathway near the

CA1/CA33 border (Fig. 1). A glass micro-electrode filled with a-CSF (resistance 1-5

MQ)) was placed in CA1 stratum radiatum (Fig. 1) to record extracellular field

potentials,, using an Axoclamp 1-D amplifier (Axon Instruments, Foster City, CA,

USA)) connected to a MacLab computer device (ADInstruments, Castle Hill ,

Australia).. Biphasic stimuli (200 /is square pulse width) were delivered, using a

PGG 4000A stimulator (Neuro Data Instruments, Delaware Water Gap, PA, USA),

att an intensity eliciting the maximal field postsynaptic potential amplitude, every

155 s throughout the experiment to monitor the viability of the slice under

baselinee conditions. GAB A release was evoked by 12 high-frequency trains (50 Hz,

3000 jiA, biphasic stimuli with alternating polarity, 200 fis square pulse width) of 3

ss duration applied at 20 s intervals, during a 4-min period (Calabresi et al., 1995).

MeasurementMeasurement of GABA release

Inn order to measure the local release of endogenous GABA, a small cannula

(fusedd silica, i.d. 180 fim, o.d. 350 ^m; Composite Metal Services, Hallow, UK)

mountedd on a micromanipulator, was placed under microscope guidance close to

thee recording electrode, approximately 100 jUm above the dendritic layers of CA1

pyramidall neurons (Fig. 1). The cannula was connected to a peristaltic pump

(modell 205S; Watson-Mariow, Falmouth, UK) set at 35 jul/min. Following a 36-

minn stabilization period, 12 consecutive 1-min fractions were collected before,

duringg and after 4 min of repetitive high-frequency stimulation. Al l experiments

weree performed in the presence of the GABA uptake inhibitor SK&F 89976-A (10

fiM),fiM), unless otherwise stated. Ca2+ dependency of electrically-stimulated GABA

releasee was determined using Ca2+-free superfusion medium with 200 11M

cadmiumm (to ascertain complete blockade of Ca2+ channels). The regulation of

GABAA release by G A B A B receptors was investigated using the G A B A B receptor

34 4

Electrically-stimulatedElectrically-stimulated GABA release in CA1 region

antagonistss saclofen (250 jiM) and CGP 52432 (100 /J.M). In these experiments, two

subsequentt 50 Hz trains of stimuli (SI and S2) separated by 15 min were applied,

eachh consisting of three 50 Hz trains of 3 s at 20-s intervals during a 1-min period.

a-CSFF containing either saclofen, or CGP 52432 was applied directly after SI and

duringg S2. Al l collected samples were diluted to a total volume of 40 fA with a-

CSFF containing 1 % (wt/vol) trichloroacetic acid and 0.25 [iM L-homoserine, as

externall standard, for GABA content determination. To avoid GABA

metabolization,, samples were kept cool (at 2°C) during the experiment by means

off a recirculation cooler (B.Braun, Melsungen, Germany) and stored at -20°C

untill GABA quantification.

Pump p

HPLC C

GAB A A

Retentio nn tim e (min ) 10

Fig.. 1. Schematic illustration of stimulation, recording and localized fluid collection

arrangements.. A hippocampal slice was submerged in a chamber with continuously flowing

oxygenatedd a-CSF. A stimulation electrode (Stim) was positioned in the Schaffer-

collateral/commissurall fibre pathway (sch/cf). A recording electrode (Rec) was placed in

CA11 stratum radiatum to monitor extracellular field responses. A glass cannula,

continuouslyy collecting extracellular fluid at 35 /d/min (indicated by the arrow), was placed

approximatelyy 100 /zm above the dendritic layers of CA1 pyramidal neurons. Collected

fractionss were analyzed by HPLC for GABA quantification.

35 5

ChapterChapter 2

QuantificationQuantification of GABA

GABAA content in the collected samples was quantified by HPLC after

p reco lumnn der ivat izat ion of a 25-//1 sample with 50 }l\ 10 mM o-

phthalaldehyde/0.44 % (vol/vol) mercaptoethanol in 100 mM Na-tetraborate (pH

10.5),, as described previously (Verhage et al., 1989). The 12.5-cm-long, 4.6-mm-

diameterr column packed with Hypersil C-18 (3 /zm particle size), was eluted

isocraticallyy with a buffer (pH 7.0) containing 0.1 M Na2HPC>4,1 mM Na2-EDTA,

0.33 % (vol/vol) tetrahydrofuran and 35 % (vol/vol) HPLC-grade methanol. The

o-phthalaldehydee derivates were detected by a Jasco (model FP-920) fluorimeter

andd evaluated by an on-line computerized data acquisition system (Gilson,

Villier ss le Bel, France). GABA levels were calculated by comparison with a

standardd sample of 0.25 fiM of this amino acid.

StatisticaiStatisticai analysis

Al ll results are expressed as means SEM. Release data were statistically

evaluatedd using the paired, or unpaired Student's f-test, as indicated. Values were

consideredd significantly different when P < 0.05.

Result s s

MeasurementMeasurement of electrically-stimulated GABA release

Underr baseline conditions, extracellular GABA levels in hippocampal CA1

regionn were detected amounting to 10.7 7 nM (Fig. 2, open circles). Local

stimulationn of 3-s 50 Hz trains applied to the Schaffer-collateral/commissural

fibress every 20 s, did not result in a significant increase of GABA release

measuredd in 1-min samples (peak value: 14.3 3.3 nM). Since the absence of an

increasee could be caused by rapid clearance of local GABA release by GABA

transporters,, the experiments were repeated in the presence of the GABA uptake

inhibitorr SK&F 89976-A (Yunger et al., 1984). Application of 10 /iM SK&F 89976-A

increasedd extracellular GABA levels under baseline conditions significantly (to

17.44 5 nM) compared to GABA levels in the absence of SK&F 89976-A (Fig. 2,

closedclosed circles; P = 0.03, unpaired Student's t-test). In the presence of 10 /iM SK&F

89976-A,, repetitive 50 Hz stimulation increased GABA release significantly

36 6

Electrically-stimulatedElectrically-stimulated GABA release in CA1 region

throughoutt the 4-min stimulation period as compared to GABA levels before 50

Hzz stimulation. The maximal increase was reached during the first min of

stimulationn (37.6 4.8 nM), declined thereafter and reached pre-stimulus values

immediatelyy after termination of the 50 Hz stimulation period (Fig. 2).

AA significant increase in electrically-stimulated GABA release could already be

observedd at a frequency of 7.5 Hz. However, at 50 Hz, this increase was more

pronouncedd and better reproducible. A single 3-s train of 50 Hz stimulation was

nott sufficient to induce a significant increase in GABA release collected during

thee 1-min sample period (data not shown).

50 0

40 0

cc 30

10 0

- 11 1 1 1 1 1 1 -1 1 1

- 3 - 2 - 1 0 1 2 3 4 5 6 6 Timee (min before/after stimulus onset)

Fig.. 2. Effect of repetitive 50 Hz stimulation on GABA release in the absence of SK&F

89976-AA (open circles, n = 5) and in the presence of 10 jjM SK&F 89976-A (closed circles, n =

11).. 50 Hz stimulus trains of 3-s duration were applied at 20-s intervals during a 4-min

periodd (indicated by the notched bar). Comparisons were made between GABA levels before

andd GABA levels during 50 Hz stimulation (*P < 0.05, **P < 0.01, paired Student's t-test).

CaCa2+2+ dependency of electrically-stimulated GABA release

Too identify whether the electrically-stimulated GABA release was of

presynapticc vesicular origin, its Ca2+ dependency was determined. In these

experiments,, the slice chamber was perfused with Ca2+-free medium containing

—— 10nM SK&F - O —— Withou t SK&F

* -- * *--$--K K K K 500 Hz

11 I ' ' I '

- $$ §

37 7

ChapterChapter 2

2000 fJ,M Cd2+ 10 min before 50 Hz stimulation. Under these conditions,

extracellularr field potentials (measured every 15 s) disappeared, usually within 5

min,, but the presynaptic fibre volley was still present, indicating that the

stimuluss was still effective. Whereas under control conditions, repetitive 50 Hz

stimulationn increased GABA release to 203 23 % of the baseline GABA level

dur ingg the first min, perfusion with Ca2+-free medium in other slices from the

samee animals, resulted in complete inhibition of this 50 Hz-stimulated GABA

releasee (Fig. 3).

500 Hz

oL.. , , M I N I M U M , , - 3 - 2 - 1 00 1 2 3 4 5 6

Timee (min before/afte r stimulu s onset )

Fig.. 3. Ca2+ dependency of electrically-stimulated GABA release. a-CSF without Ca2+

andd with 200 fxM Cd2+ was applied 10 min before stimulation (closed circles, n = 6). Control

experimentss were performed in other slices from the same animals using a-CSF with 2 mM

Ca2++ and without Cd2+ (open circles, n = 6). All experiments were performed in the presence

off 10 nM SK&F 89976-A. GABA release was expressed as percentage of mean release under

baselinee conditions before 50 Hz stimulation. 50 Hz stimulus trains of 3-s duration were

appliedd at 20-s intervals during a 4-min period (indicated by the notched bar). Comparisons

weree made between GABA levels before and GABA levels during 50 Hz stimulation ( P <

0.05,, **P < 0.01, paired Student's t-test).

—— Withou t Calciu m — O —— Contro l

38 8

Electrically-stimulatedElectrically-stimulated GABA release in CA1 region

LocalLocal restriction of electrically-stimulated GABA release

Thee local restriction of the electrically-evoked GABA release by stimulation of

thee Schaffer-collateral/commissural fibres to the CA1 region, when using a

cannulaa above the CA1 stratum radiatum, was investigated by measuring GABA

releasee simultaneously by a second cannula placed above the dentate gyrus

region,, at about 1 mm distance from each other. Because the electrically-

stimulatedd GABA release observed in the previous experiments was maximal

duringg the first min of repetitive 50 Hz stimulation (see Fig. 2), the total duration

off the stimulus was 1 nun. Whereas GABA release upon this stimulation of the

Schaffer-collateral/commissurall fibres was increased in the CA1 region, no

changess in extracellular GABA levels were observed in the dentate gyrus region

(Fig.. 4).

40-, ,

30--

ChapterChapter 2

RegulationRegulation of electrically-stimulated GAB A release by GABAB receptors receptors

Too investigate whether electrically-stimulated GABA release is regulated by

presynapticc G A B A B receptors, we used the broad G A B A B receptor antagonist

saclofenn (Kerr et al., 1989) and the selective high-affinity G A B A B autoreceptor

antagonistt CGP 52432 (Lanza et al., 1993). These experiments were performed

usingg two sets of 50 Hz trains of stimuli (SI and S2), applied sequentially at an

intervall of 15 min, first in the absence (SI) and then in the presence (S2) of either

2500 jxM saclofen, or 100 [iM CGP 52432 (see Materials and methods). Using these

concentrations,, a maximal increase in GABA release was observed. GABA release

wass measured during SI and S2. The S2/S1 ratio of GABA release of 1.05 0.02 in

controll slices, was increased to 1.36 0.09 by application of saclofen (Fig. 5).

Applicationn of CGP 52432 increased the S2/ SI ratio to an equal value (of 1.33

1,6-, ,

DD Contro l SS Saclofe n

Fig.. 5. Effects of 250 /xM saclofen and 100 fiM CGP 52432 on 50 Hz-stimulated GABA

release.. Experiments were performed using two high-frequency stimuli (SI and S2) separated

byy 15 min, each consisting of three 50 Hz trains of 3 s applied at 20-s intervals during a 1-

minn period. a-CSF containing either saclofen, or CGP 52432 was applied directly after SI

andd during S2. Data are expressed as the mean S2/S1 ratio SEM of GABA release

measuredd during SI and S2. Comparisons were made between S2/S1 in the absence (control,

nn = 13) and S2/S1 in the presence of either saclofen (n = 6), or CGP 52432 (n = 9) (**P <

0.001,, unpaired Student's Mest).

40 0

Electrically-stimulatedElectrically-stimulated GABA release in CA1 region

0.05)) as induced by saclofen, indicating G A B A B autoreceptor-mediated

disinhibitionn of GABA release during high-frequency stimulation.

Discussio n n

Inn this study, a biochemical method to measure local presynaptic GABA

releasee locally in the CA1 region of hippocampal slices was presented. The main

findingss were: (1) Local high-frequency electrically-stimulated GABA release

fromm the CA1 region can be measured when GABA uptake is blocked. (2) This

stimulatedd GABA release is mainly derived from a presynaptic vesicular

transmitterr pool and is restricted to the CA1 region. (3) The tetanically-stimulated

GABAA release is regulated by presynaptic G A B A B autoreceptors.

Thiss study shows, for the first time, that local presynaptic GABA release from

thee hippocampal CA1 region can be measured biochemically under rather

physiologicall conditions. In several biochemical studies, depolarization with

highh K+ was used to study GABA release from hippocampal tissue. However,

suchh a massive stimulus wil l drastically recruit GABA from its different

releasablee pools from neurons as well as glial cells, rather than triggering limited

amountss of GABA release from locally excited GABAergic nerve endings.

Therefore,, K+-induced GABA release represents total capacities of releasable

pools,, rather than actual GABA release. To illustrate this, we compared GABA

releasee evoked by high-frequency electrical stimulation with that by 50 mM K+

stimulation.. Using this K+ stimulation, both Ca2 +-dependent (vesicular) and

Ca2+- independentt release of GABA release via reversal of GABA transporters

occurss (Attwell et ah, 1993; Belhage et al., 1993; Richerson & Gaspary, 1997). In

contrast,, the electrically-stimulated GABA release was probably from presynaptic

vesicularr origin only, since it was completely suppressed under Ca2 +-free

conditionss (Fig. 3). The exocytotic fraction of the K+-stimulated GABA release was

aboutt 400 nM (not shown), i.e. 11-fold higher than the peak value of 37.6 nM

observedd during local high-frequency stimulation (Fig. 2).

Otherr studies, using the same method as we applied, have shown increases in

thee levels of several amino acids, but not GABA, from hippocampal slices upon

Schaffer-collateral/commissurall fibre stimulation (Spencer et al., 1981; Roisin et

al.,, 1991; Klancnik et al., 1992). However, in these studies high-frequency

stimulationn (10-50 Hz) was applied continuously for several minutes, which is

ratherr unphysiological and may exhaust the readily releasable pool of GABA.

41 1

ChapterChapter 2

Klancnikk et al. (1992), also applied very brief tetanic stimulus trains (600 ms, 100

Hz)) which produced LTP, but changes in aspartate, glutamate or GABA release

weree not observed. They speculated that the absence of an effect might be

explainedd by efficient uptake of these amino acids without being monitored by

thee cannula above the slice. The fact that in our study enhanced GABA release

couldd be observed only in the presence of SK&F 89976-A, indicates that local

GABAA release from the slice was indeed efficiently cleared by its transporters, and

thatt inclusion of a GABA uptake inhibitor is required to monitor electrically-

stimulatedd GABA release by the cannula. Surprisingly, an increased release of

aspartatee and glutamate upon tetanic stimulation, even in the presence of the

glutamate/aspartatee uptake inhibitor L-£raws-pyrrolidine-2,4-dicarboxylate (L-

frons-PDC),, was not detected (results not shown). Possibly, the increase in local

releasee of these amino acids, occurring during the 3-s tetanus trains only, is

maskedd by spontaneous release evoked by L-trans-PDC acting as a substrate of the

glutamate/aspartatee transporters as well (Madl & Burgesser, 1993; Griffit h et al.,

1994). .

Thee study of Corradetti et al. (1983) demonstrated enhanced GABA release

uponn Schaffer-collateral/commissural fibre stimulation. However, stimuli (at 2

Hz)) were applied continuously for 5 min. Moreover, release was not measured

locallyy since superfusion fluid from the whole hippocampal slice was collected. It

iss important to restrict GABA release measurements to hippocampal sub-regions,

sincee a diversity of GABAergic functions in the different regions has been

describedd (see for example Lopes da Silva et al., 1995). The absence of an effect on

GABAA release in the dentate gyrus upon stimulation of the Schaffer-

collateral/commissurall fibres observed in our study, indicates that the evoked

releasee we observed was restricted to the CA1 region. Calabresi et al. (1995)

observedd increased GABA release from striatal slices using a stimulation protocol

similarr to ours (100 Hz, three trains, 3 s duration, 20 s intervals).

Interestingly,, the method allows also measurement of regulatory mechanisms

off GABA release. The enhancement of GABA release by CGP 52432, indicates

activee regulation by presynaptic GABA R autoreceptors during high-frequency

stimulation.. This result is in line with electrophysiological studies indicating

regulationn of GABA release by GABA R autoreceptors at higher frequencies

(Daviess & Collingridge, 1993; Mott et al., 1993). The finding that saclofen, which

doess not discriminate between different GABA R receptor subtypes (Misgeld et al.,

1995),, has similar effects on GABA release as CGP 52432 strongly suggests that the

electrically-stimulatedd GABA release in the CA1 region of hippocampal slices is

42 2

Electrically-stimulatedElectrically-stimulated GABA release in CA1 region

primarilyy regulated by presynaptic G A B A B autoreceptors. In addition, application

off 100 fiM of the G A B A B heteroreceptor antagonist CGP 35348 (Olpe et al., 1990),

whichh has been indicated to antagonize postsynaptic G A B A B receptors as well at

high-affinityy (Deisz et al., 1997), had no effect on electrically-stimulated GABA

releasee (data not shown). Our study shows, for the first time, that local

presynapticc GABA release in hippocampal slices is regulated by G A B A B

autoreceptors.. Regulation of GABA release by CGP 52432 has been shown earlier,

however,, in cortex synaptosomes using high K+ stimulation (Lanza et al., 1993;

Bonannoo et al., 1997). Waldmeier et al. (1993, 1994) observed regulation of

electrically-stimulatedd GABA release in cortical slices by different G A B A B

receptorr antagonists. However, GABA release in those studies were evoked by

massivee electrical stimulation of the whole slice chamber, instead of local fibre

stimulationn as applied in our experiments.

Inn conclusion, this method allows direct measurement of electrically-evoked

GABAA release, as well as its presynaptic receptor-mediated regulation in the CA1

regionn of hippocampal slices. The method can be useful to investigate alterations

inn GABA release in processes of synaptic plasticity in the hippocampus, such as

epilepsy. .

Acknowledgement s s

Thee authors are indebted to the Central Institute of Feeding Research (TNO,

Zeist,, The Netherlands) for packing HPLC columns. This work was financially

supportedd by the Netherlands Organization for Scientific Research (NWO, grant

903-42-091). .

43 3

44 4