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Taurine, a sulfonated b-amino acid is involved in important

physiological functions participating in cell volume

regulation, the maintenance of structural integrity of cell

membranes and intracellular calcium homeostasis

(Huxtable, 1992; Oja & Saransaari, 1996; Lang et al. 1998;

Haussinger et al. 2000). Taurine is released in large

quantities in the brain under hyposmotic conditions,

energy deprivation and by cell depolarization (Deleuze etal. 1998; Bockelmann et al. 1998; Colivicchi et al. 1998). Its

release occurs through osmo-sensitive anion channels

(Strange & Jackson, 1995; Bres et al. 2000; Hussy et al.2001) or through the taurine transporter (TAUT) working

in reverse mode (Saransaari & Oja, 1999).

TAUT located on the surface membranes of glia and

neurones accumulates taurine intracellularly, which is of

particular importance for neurones unable to synthesize

taurine (Brand et al. 1993). Recently, two isoforms of

TAUT, which are identical except for their C-terminal

sequences, were mapped in the rat brain (Pow et al. 2002).

In accordance with previous studies in rat (Smith et al.1992) and mouse (Vinnakota et al. 1997) the TAUT1

protein was found mainly in the neurons of the retina and

cerebellum, where its distribution correlated with the

accumulation of high levels of taurine. Prominent TAUT2

staining was seen in the hippocampus and the cerebellum

(Pow et al. 2002), mainly in non-neuronal cells and not

accompanied by [3H]taurine accumulation. However,

TAUT2 mRNA, originally sequenced from mouse brain

(Liu et al. 1992), occurs in high concentrations in the

striatum, the corpus callosum and the brain stem. Striatal

projecting neurons also accumulate [3H]taurine (Clarke etal. 1983). Disturbances in excitatory transmission to the

striatum are deemed responsible for the symptoms

observed in hepatic encephalopathy (Saransaari et al. 1997).

The reduced content of extracellular taurine due to

intracellular accumulation is associated with encephalo-

pathy caused by liver failure (Hilgier et al. 1999).

A taurine-evoked long-lasting potentiation of synaptic

transmission in hippocampal (Galarreta et al. 1996b;

del Olmo et al. 2000) and cortico-striatal slices (Chepkova

et al. 2002) has previously been reported and the taurine

uptake mechanism was suggested to be responsible for this

phenomenon. The potentiation followed an initial

depression, which was attributed to the direct activation of

GABAA receptors by taurine. These two phenomena could

occur independently of each other (Galarreta et al. 1996b).

While the role of taurine as a ligand of inhibitory iono-

tropic receptors is well studied (Haas & Hosli, 1973; Hussy

et al. 1997; Sergeeva & Haas, 2001), the impact of TAUT

activation on neuronal excitability is unclear. Taurine

transport could modulate neuronal excitability, synaptic

transmission and plasticity through its electrogenic nature

(Ramamoorthy et al. 1993; Vinnakota et al. 1997), especially

in cases of close location and coordinated activation with

Taurine-induced long-lasting enhancement of synaptictransmission in mice: role of transportersO. A. Sergeeva*, A. N. Chepkova†, N. Doreulee*, K. S. Eriksson*, W. Poelchen*, I. Mönnighoff*, B. Heller-Stilb*, U. Warskulat‡, D. Häussinger‡ and H. L. Haas*

Departments of *Neurophysiology and ‡Gastroenterology, Hepatology and Infectiology, Heinrich-Heine-Universität, PO Box 101007,D-40001 Düsseldorf, Germany, and †Brain Research Institute, Russian Academy of Medical Sciences, Moscow 103064, Russia

Taurine, a major osmolyte in the brain evokes a long-lasting enhancement (LLETAU) of synaptic

transmission in hippocampal and cortico-striatal slices. Hippocampal LLETAU was abolished by the

GABA uptake blocker nipecotic acid (NPA) but not by the taurine-uptake inhibitor guanidinoethyl

sulphonate (GES). Striatal LLETAU was sensitive to GES but not to NPA. Semiquantitative PCR

analysis and immunohistochemistry revealed that taurine transporter expression is significantly

higher in the striatum than in the hippocampus. Taurine transporter-deficient mice displayed very

low taurine levels in both structures and a low ability to develop LLETAU in the striatum, but not in

the hippocampus. The different mechanisms of taurine-induced synaptic plasticity may reflect the

different vulnerabilities of these brain regions under pathological conditions that are accompanied

by osmotic changes such as hepatic encephalopathy.

(Resubmitted 27 April 2003; accepted after revision 12 May 2003; first published online 24 June 2003)

Corresponding author O. A. Sergeeva: Heinrich-Heine-Universität, Physiology II, PO Box 101007, D-40001 Düsseldorf,Germany. Email: olga.sergeeva@uni-duesseldorf.de

J Physiol (2003), 550.3, pp. 911–919 DOI: 10.1113/jphysiol.2003.045864

© The Physiological Society 2003 www.jphysiol.org

O. A. Sergeeva and A. N. Chepkova contributed equally to this work.

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synapses. To clarify the role of TAUT in the taurine-

induced long-lasting enhancement of synaptic transmission

(LLETAU) we have now studied this phenomenon in the

striatum and hippocampus of TAUT knockout (KO) mice

(Heller-Stilb et al. 2002) and after pharmacological

inhibition of taurine transport. We report a pronounced

deficit of taurine-evoked synaptic enhancement in the

striatum, but not in the hippocampus of TAUT KO mice

and different sensitivities of striatal and hippocampal

LLETAU to taurine uptake inhibitors.

METHODSField potential recordingsGenotyped 6- to 11-week-old TAUT KO (n = 11) and wild-type(WT, n = 10) mice, the male and female offspring of the sameheterozygous breeding pairs, and male C57BL/6 mice of the sameage (n = 22) were used for electrophysiological experiments. Allexperiments were conducted in compliance with German Lawand with the approval of Bezirksregierung Duesseldorf. Theanimals were decapitated and the brains rapidly removed, placedin ice-cold Krebs–Ringer solution and cut into horizontal slices,400 mm thick, using a Vibroslicer (Campden Instruments). Theslice preparations included the neostriatrum, the neocortex andthe hippocampus (Fig. 1A). After 2–3 h preincubation at roomtemperature a single slice was transferred to a recording chamberand submerged in a continuously flowing (1.5 ml min_1) mediumwarmed to 32–33 °C. Both preincubation and perfusion mediacontained (mM) 124 NaCl, 3.7 KCl, 1.24 NaH2PO4, 1.3 MgSO4, 2.0CaCl2, 26 NaHCO3, glucose 20 and were continuously saturatedwith a 95 % O2–5 % CO2 mixture.

Low-resistance glass micropipettes filled with perfusion mediumwere used to record field potentials in the neostriatum and in thestratum radiatum of the CA1 area of the hippocampus. A bipolarNi–Cr stimulation electrode was placed on the white matterbetween the cortex and the neostriatum to stimulate the cortico-striatal axons, and on the hippocampal radial layer to stimulateSchaffer collaterals. The positions of stimulating and recordingelectrodes are indicated on the schematic picture of a horizontalslice (Fig. 1A). Constant-voltage pulses of 80 ms duration wereapplied every 20 s. Stimulus intensity was adjusted to induce a half-maximal response. Signals were amplified, digitized at 10 kHz,and recorded on a PC using pCLAMP software (Axon Instruments).

The standard experimental protocol included 30 min controlrecording, 30 min perfusion with 10 mM taurine and a 60 minwashout period. Taurine (Sigma) was dissolved in the perfusionmedium containing 10 mM glucose instead of 20 mM glucose tomaintain the constant osmolarity and was applied by switching thetwo-tap perfusion system to the reservoir with taurine-containingmedium. The osmolarity of solutions was controlled with anosmometer (Knauer, Berlin, Germany). In control experiments,glucose (10 mM) substitution with sucrose did not affect fieldpotentials. Nipecotic acid was obtained from Sigma andguanidinoethyl sulphonate was from Toronto Research Chemicals.

Fifteen consecutive responses (5 min recording) were averagedoff-line to generate one data point. The characteristic fieldpotential evoked by cortical white matter stimulation consisted oftwo negative potentials: the first (N1) reflecting a fibre potentialand direct activation of medium spiny neurons and the second(N2) being a synaptically induced wave. The amplitude of the N2

peak evoked by cortical white matter stimulation and the fieldEPSP slope were measured in the striatum and the hippocampus,respectively. All values were normalized to the mean value over the30 min control period. The data were expressed as means ± S.E.M.and analysed statistically using Student’s t test (two-tailed) andFisher’s exact probability test.

Real-time RT-PCRTotal cellular mRNA was isolated from the hippocampus and thedorsal striatum of either side using a mRNA isolation kit (PharmaciaBiotech) according to the manufacturer’s protocol. Total mRNAwas eluted from the matrix with 200 ml of RNase-free water. Forreverse transcription, 8 ml of eluted mRNA was added to 7 mM

reagent mixture prepared according to the protocol of the ‘firststrand cDNA synthesis kit’ (Pharmacia Biotech). After incubationfor 1 h at 37 °C the reverse transcription reaction was stopped byfreezing at _20 °C. The reverse-transcription reactions were notnormalized to contain the equivalent amounts of total mRNA. ThePCR was performed in a PE Biosystems GeneAmp 5700 sequencedetection system using the SYBR green master mix kit. Each reactioncontained 2.5 ml of the 10xSYBR green buffer, 200 nM each of dATP,dGTP and dCTP, 400 nM dUTP, 2 mM MgCl2, 0.25 units of uracilN‚-glycosylase, 0.625 units of Amplitaq Gold DNA polymerase,10 pM forward and reverse primers, 5 ml of 1:4 diluted cDNA, andwater to 25 ml. Primers used for the semiquantitative analysis were asfollows: b-actin forward: 5‚-CGT GAA AAG ATG ACC CAG ATCATG TT-3‚; b-actin reverse: 5‚-GCT CAT TGC CGA TAG TGATGA CCT G-3‚; TAUT forward: 5‚-GAA AGA CTT CCA CAA AGACAT CC-3‚; TAUT reverse: 5‚-GTA CTG GCC TAT GAT GAC CTCC-3‚. The reactions were performed in MicroAmp 96-well plates orin optical tubes capped with MicroAmp optical caps. The reactionswere incubated at 50 °C for 2 min to activate uracil N‚-glycosylase,and then for 10 min at 95 °C to inactivate the uracil N‚-glycosylaseand activate the Amplitaq Gold polymerase, followed by 40 cycles of15 s at 95 °C, 1 min at 60 °C. The PCR reactions were subjected to aheat dissociation protocol (PE Biosystems 5700 software). Followingthe final cycle of the PCR, the reactions were heat denaturated over a35 °C temperature gradient at 0.03 °C s_1 from 60 to 95 °C. Each PCRproduct showed a single peak in the denaturation curves. Theidentity of PCR products with the known cDNA sequences wasdetermined as described before (Sergeeva & Haas, 2001). Standardcurves for real-time PCR protocols with both primer pairs obtainedwith sequential dilutions of one cDNA sample (down to 1:1000)were found to be optimal (linear regression coefficients were 0.99and 0.97 for the b-actin and TAUT respectively, P < 0.01).

The real-time PCR data were plotted as the fluorescence signalversus the cycle number. The PE Biosystems 5700 sequencedetection system software calculates the DRn (Reporter,normalized) using the equation:

DRn = (Rn+) _ (Rn

_),

where Rn+ is the fluorescence signal of the product at any given

time and Rn_ is the fluorescence signal of the baseline emission

during cycles 6–15. An arbitrary threshold was set at the midpointof logDRn versus cycle number plot. The Ct value is defined as thecycle number at which the DRn crosses this threshold. The changein taurine transporter (TAUT) cDNA (target gene) relative to theb-actin endogenous control was determined by:

Fold change = 2_DDCt,

where

DDCt = (CtTarget _ CtActin)time x _ (CtTarget _ CtActin)time 0.

O. A. Sergeeva and others912 J Physiol 550.3

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Time x is any time point and time 0 represents the 1 w expressionof each gene under conditions of serum starvation. Relativequantification of gene expression using the 2_DDCt methodcorrelated with the absolute gene quantification obtained instandard curves (Winer et al. 1999).

ImmunohistochemistryHorizontal slices of 0.5–1 mm thickness containing the striatumand the hippocampus were fixed for 8 h in phosphate-buffered4 % paraformaldehyde (pH 7.4) at 4 °C, cryoprotected in sucrose,cryosectioned at 24 mm thickness, and mounted on gelatin-coatedslides. All antibody incubations and washes were carried out inphosphate-buffered saline with 0.25 % Triton X-100 (pH 7.4) andall antibody solutions contained 2 % normal swine serum. Todetermine the localization of the taurine transporter we used arabbit anti-taurine transporter serum (Tau11-A, DPC Biermann,Bad Nauheim, Germany) diluted 1:500–1:1000. Primary anti-serum was applied to the sections for 24 h at 4 °C, and thefollowing steps were performed at room temperature. After theincubation with primary antibody, the slides were incubated witha biotinylated swine-anti-rabbit serum (1:200; DAKO, Hamburg,Germany) for 2 h and then with an ABC complex (1:500;Vectastain elite, Vector Laboratories, Burlingame, CA, USA) for2 h. The immunoreactivity was then visualized by an 8–12 minincubation in a solution of 0.03 % 3,3‚-diaminobenzidine tetra-hydrochloride, 0.015 % H2O2, and 0.1–0.2 % NiCl2 in Tris-HCl(pH 7.6), which yielded a black reaction product. When theprimary antiserum was replaced with normal rabbit serum, thestaining was absent.

Determination of taurine levelsDifferent brain structures were dissected under a microscope andthe freshly removed tissues were frozen in liquid nitrogen andthen stored at _70 °C. Frozen tissue was homogenized andweighed five times without thawing. Proteins were removed byincubation in 10 % sulfosalicylic acid on ice for 1 h. Aftercentrifugation for 10 min at 21 000 g, the lipids were extractedfrom the supernatant with dichloromethane. Plasma was added toan equal amount of 10 % sulfosalicylic acid. Taurine content wasmeasured in a BioChrom20 amino acid analyser (Amersham-Pharmacia Biotech, Freiburg, Germany).

RESULTSTaurine transport and LLETAU

Bath application of taurine (10 mM for 30 min) to hippo-

campal and cortico-striatal slices from control (WT or

C57BL/6) mice caused an initial depression of synaptic

field responses followed by their long-term enhancement

(Figs 1B, 2 and 3). The initial depression was previously

attributed to activation of GABAA receptors in the hippo-

campus (Galarreta et al. 1996b) and both GABAA and

glycine receptors in the striatum (Chepkova et al. 2002).

After taurine withdrawal the field potentials recovered

their amplitudes and, in the vast majority of both hippo-

campal and cortico-striatal slices, they developed a long-

lasting enhancement, LLETAU. The data on taurine-induced

Synaptic potentiation by taurineJ Physiol 550.3 913

Figure 1. Illustrations of a horizontal brain slice (A) and long-lasting enhancement ofhippocampal synaptic transmission (B)The time course of CA1 field potential amplitudes (fEPSP) is illustrated in B. Taurine-induced long-lastingenhancement (LLETAU) of synaptic field responses in control (•) is unchanged in the presence of the taurineuptake inhibitor guanidinoethyl sulphonate (GES, ª). Times of taurine (TAU, 10 mM) and GES applicationsare indicated by horizontal bars. Abbreviations in A: CX, cortex; CS, corpus striatum; GP, globus pallidus;Th, thalamus; HPC, hippocampus.

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enhancement of the CA1 field EPSP and cortico-striatal

postsynaptic responses are summarized in Table 1 and

illustrated in Figs 1, 2 and 3.

Guanidinoethyl sulphonate (GES) is a competitive inhibitor

of high-affinity taurine uptake. GES (1 mM) applied 10 min

before and for 30 min together with the application of

taurine did not affect the development of LLETAU in the

Schaffer collateral–CA1 hippocampal pathway. In contrast

to cortico-striatal slices (Chepkova et al. 2002), GES exerted

no effects on either the occurrence (5 of 5 slices) or the

magnitude (147.8 ± 11.9 % of baseline, n = 5, vs.144.5 ± 7.6 %, n = 14, in the control) of LLETAU in

hippocampal slices from C57BL/6 mice (Fig. 1B).

The LLETAU phenomenon was significantly less pronounced

in the striatal, but not in the hippocampal slices from TAUT

KO mice (Figs 2 and 3). A taurine-induced enhancement

of cortico-striatal field potentials occurred in only 8 of 20

slices from the KO mice (P = 0.0004, Fisher’s exact

probability test) and tended to have a lower magnitude

(not significant, P = 0.076, Student’s t test) than in slices

from WT mice (Table 1). TAUT KO and WT mice did not

significantly differ in the occurrence (P = 0.27, Fisher’s exact

probability test) and magnitude (P = 0.318, Student’s ttest) of LLETAU in hippocampal slices (Table 1, Fig. 2),

although both parameters were slightly lower in TAUT KO

than in WT mice. In hippocampal slices from TAUT KO

mice the initial depression was less pronounced than in

those from WT mice (82.45 ± 2.42 % of the baseline versus59.0 ± 6.5 % in WT, P < 0.01). No difference was seen in

the initial depression between TAUT KO (to 40.8 ± 4.4 %

of the baseline, n = 20) and WT (to 39.4 ± 5.1 %, n = 18)

in the striatum (Fig. 3).

O. A. Sergeeva and others914 J Physiol 550.3

Figure 2. Taurine evokes long-lastingenhancement of synaptic transmission (LLETAU)in hippocampal slices from both wild-type (WT)and TAUT knockout (KO) miceA and B, time courses of changes in the field EPSP(fEPSP) slope function in the hippocampus (CA1).Diagrams summarize data obtained in 16 WT and 18TAUT KO mice. Taurine (TAU, 10 mM) was applied forthe time periods indicated by horizontal bars.C, representative field potentials in WT mouse. Tracesare taken from a single experiment before (upper)during (middle) and 1 h after (lower) taurine exposure.Each trace is an average of 15 individual field potentials.D, averaged input–output plots, obtained fromhippocampal slices of 10 WT and 8 TAUT KO mice.Data points in each experiment were normalized on themaximal response amplitude.

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Basal synaptic functions in hippocampal and cortico-striatal

slices as measured by input–output relations, and peak

response amplitudes were not significantly different between

WT and KO mice. The striatal N2 amplitudes were identical

(WT, 1.45 ± 0.08 mV, n = 8, and KO, 1.45 ± 0.11 mV,

n = 10, P = 0.99) The maximal slopes of hippocampal field

EPSPs (fEPSPs), subthreshold for pop-spike generation did

not differ significantly (0.60 ± 0.06 mV ms_1, n = 10, and

0.51 ± 0.03 mV ms_1, n = 8, in WT and KO, respectively,

P = 0.22). The input–output curves for hippocampal and

striatal slices are presented in Figs 2 and 3.

GABA transporter antagonist prevents LLETAU inhippocampus but not in striatumUnder conditions of high extracellular concentration,

taurine can be taken up by GABA transporters (Liu et al.1993). When nipecotic acid (NPA, 1 mM), a broad-spectrum

GABA transporter (GAT) antagonist, was applied 10 min

before and together with taurine, it prevented LLETAU

(Fig. 4, 102.6 ± 4.9 %, n = 6) in hippocampal slices from

C57BL/6 mice. In these slices, the initial depression reached

50.4 ± 8.6 %, n = 6 vs. 69.0 ± 6.0 %, n = 15 (taurine alone,

difference not significant). In cortico-striatal slices pretreated

with NPA the LLETAU magnitude (174.5 ± 18.8 %, n = 4) did

not significantly differ from the control (149.5 ± 6.0 %,

n = 11, P = 0.1), while the initial depression was significantly

deeper (11.7 ± 2.0 % of baseline, n = 4, vs. 55.6 ± 5.3,

n = 12 in the control, P < 0.01; Fig. 4).

In hippocampal slices from TAUT KO mice, 10 mM taurine

in the presence of 1 mM NPA also caused a significantly

deeper initial depression than in the control (to

43.9 ± 11.9 % of the baseline, n = 4, vs. 81.5 ± 5.1 %, n = 5,

in the control, P < 0.01) and only a transient post-taurine

Synaptic potentiation by taurineJ Physiol 550.3 915

Figure 3. LLETAU of synaptic transmissionin striatal slices from WT but not fromTAUT KO miceA and B, time courses of changes in the secondnegative component of the field (N2). Diagramssummarize time courses of postsynapticchanges in WT (n = 18) and TAUT KO (n = 20)mice. Taurine (10 mM) was applied for the timeperiods indicated by the horizontal bars.C, representative field potentials in WT mice areinserted; traces are taken from a singleexperiment before (upper) during (middle) and1 h after (lower) taurine exposure. Each trace isan average of 15 individual field potentials.D, averaged input–output plots from striatalslices of 8 WT and 10 TAUT KO animals. Datapoints in each experiment were normalized tothe maximal response amplitude.

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potentiation of field responses (Fig. 4). LLETAU in the

control slices peaked at 20–30 min after taurine with-

drawal and remained at this level to the end of recording

(up to 90 min of washout), whereas in slices exposed to

taurine in combination with NPA, field responses returned

to the baseline during this period (Fig. 4).

NPA, applied for 20 min at 10 mM, induced an almost

complete depression of field potentials in the hippo-

campus (to 5.2 ± 2.9 % of baseline, n = 3); partial recovery

was reached after 1 h of washout (87.4 ± 4.5 %).

TAUT distribution in the hippocampus and thestriatumSemiquantitative real-time PCR revealed TAUT mRNA

levels to be 2.5 times higher in the dorsal striatum than in the

hippocampus. The values, 2_DDCt, were 5.0 ± 0.9, n = 10, for

striatum, and 2.0 ± 0.4, n = 7, for the hippocampus;

P < 0.02. The strongest immunostaining with the TAUT

antibody was obtained in the dorsal part of the striatum,

followed by a narrow region in the hippocampal CA3 area

containing the mossy fibre endings. The hippocampus, the

globus pallidus and the thalamus were stained more weakly

than the dorsal striatum (Fig. 5). Due to strong staining of

O. A. Sergeeva and others916 J Physiol 550.3

Figure 4. The GABA transporter antagonist nipecotic acid (NPA) blocks LLETAU inhippocampus, but not in striatumTime courses of changes in the second negative component of the field potential in striatum and the fieldEPSP (fEPSP) slope in CA1 hippocampus. The time periods of taurine (TAU, 10 mM) and NPA (1 mM)application are indicated by the open bars and the filled, thinner bars, respectively. Representative fieldpotentials from striatum of C57BL/6 are inserted at the right.

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the neuropil, possibly of dendrites, no somatic staining could

be clearly discerned in either structure, even under higher

magnification.

Taurine content in discrete brain areasTaurine concentrations were measured in the brains of three

animal groups: TAUT KO, WT and heterozygous (HT)

mice. They were markedly reduced in all five areas studied in

TAUT KO mice (Table 2) to between 2 % (hippocampus)

and 10 % (brain stem) of the level in WT animals. The

hippocampus and the dorsal striatum contained similar

amounts of taurine in WT and KO animals, at high and low

levels, respectively. However, the taurine contents in these

two structures differed significantly in HT mice: while the

striatum of WT and HT mice had similar taurine contents, it

was considerably reduced in the hippocampus of HT

animals.

DISCUSSIONOur experiments with TAUT KO mice have revealed that

high-affinity taurine transport considerably contributes to

a taurine-induced long-lasting enhancement (LLETAU) of

cortico-striatal transmission but is much less important in

this respect in the Schaffer collateral–CA1 pathway. The

role of TAUT in synaptic modulation is difficult to study in

the absence of reliable pharmacological tools. The only

available antagonist of taurine transport, GES, manifests a

number of additional activities such as inhibition of GABA

(Li & Lombardini, 1990) and creatine (Dai et al. 1999)

transporters and direct interaction with GABAA receptors

(Mellor et al. 2000). We have shown previously that GES is

a glycine receptor antagonist (Sergeeva et al. 2002), that

glycine receptor activation is necessary for the striatal

LLETAU, and that LLETAU is sensitive to GES (Chepkova etal. 2002).

In the CA1 area of the hippocampus, where the involve-

ment of taurine uptake in the long-lasting enhancement of

responses after exposure to taurine has been suggested

(Galarreta et al. 1996a,b), we found no significant difference

in LLETAU between WT and TAUT-deficient mice. Thus,

despite an apparent similarity in the time course and

magnitude of striatal and hippocampal LLETAU, the

mechanisms of these phenomena as well as the mecha-

nisms of taurine uptake in these structures may be

different.

Immunostaining and semiquantitative real-time RT-PCR

revealed that levels of TAUT mRNA and TAUT protein in

the hippocampus are lower than in the striatum. Our

staining was done with TAUT antiserum, analogous to

TAUT1 (Pow et al. 2002). In contrast to the sagittal

sections from rats (Pow et al. 2002) our horizontal mouse

slices displayed intense staining of the CA3 stratum

lucidum. A prominent role of taurine in the CA3 region

has recently been demonstrated (Mori et al. 2002). The

absence of this protein from CA1 in our horizontal mouse

brain sections is in keeping with the electrophysiological

data, indicating that LLETAU in this brain area is TAUT

independent.

The hippocampus and striatum of WT animals did not

differ in their taurine content, in agreement with previous

findings (Palkovits et al. 1986). Heterozygous mice, on the

other hand, displayed a higher taurine content in the

striatum, where it was indistinguishable from the control

Synaptic potentiation by taurineJ Physiol 550.3 917

Figure 5. Immunostaining for the TAUTproteina, the strongest staining is seen in the neuropil of thestriatum and in a narrow band in the hippocampalCA3 region. Scale bar, 1 mm. Higher magnifications:hippocampal CA3 (b) and dorsal neostriatal (c)fields. No stained somata can be identified withcertainty. Scale bars, 50 mm.

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level, than in the hippocampus, where the taurine content

was 1.8 times lower. The ceiling of taurine accumulation

can explain the higher taurine level seen in the hetero-

zygous but not in WT mice.

Taurine and GABA transporters differ much more in their

sensitivity to GABA than to taurine (Sivakami et al. 1992);

therefore, 10 mM taurine can activate all of them equally.

Among the four known GABA transporters, GAT-1 and

GAT-3 (called GAT-4 in mouse) are neuronal transporters

(Liu et al. 1993; Borden et al. 1995). An in situ hybridiz-

ation study (Durkin et al. 1995) revealed a high expression

of GAT-1 and a moderate expression of GAT-3 in the

pyramidal layers of rat CA1 and CA3, while only very low

staining for GAT-1 was observed in the striatum. This is in

keeping with our failure to block LLETAU in the striatum

with the GAT antagonist NPA. On the other hand,

hippocampal LLETAU turned out to be very sensitive to

NPA in both C57BL/6 and TAUT KO mice. NPA is a

substrate for the GABA transporter (Solis & Nicoll, 1992)

and when taken up causes the release, via heteroexchange,

of GABA from the cytoplasm. Our experiments with NPA

(10 mM) on hippocampal slices indicate that the activation

of GABA transporters causing GABA release is not sufficient

for eliciting LLE, but intracellular accumulation of taurine

rather than activation of transporters seems to be critical

for the development of hippocampal LLETAU.

We observed a smaller initial depression during taurine

application in TAUT KO mice and a dramatic augment-

ation of this depression by NPA in the hippocampus. The

exact reason for the lower level of taurine-induced

depression in the hippocampus of TAUT KO mice is at the

present unclear; down-regulation of GABAA receptors in

this KO model may be responsible.

The function of the TAUT2 isoform in CA1 is obscure: its

distribution does not match the extensive [3H]taurine

accumulation into neuronal and glial elements in all

hippocampal regions (Pow et al. 2002), leading to the

suggestion that as yet unknown taurine transport systems

may exist in the hippocampus. However, our data do not

support this conclusion. The KO mice in our study were

deficient in TAUT1 and TAUT2 as exon 1 was deleted

(Heller-Stilb et al. 2002). They showed a massive loss of

taurine in the hippocampus, indicating that GABA trans-

porters, although responsible for LLETAU, cannot compensate

for the missing TAUT function.

It has been suggested that hippocampal LLETAU is initiated

by taurine accumulation (Galarreta et al. 1996b) and

depends on low-voltage-activated calcium channels

(LVACC) (del Olmo et al. 2000). Hippocampal LLETAU

was partially occluded by tetanus-evoked long-term

potentiation (LTP) and was reversed by low-frequency

stimulation suggesting shared mechanisms with LTP

(del Olmo et al. 1998, 2000). Striatal cells show the presence

of LVACC of a different subtype to that in the hippo-

campus (McRory et al. 2001), which can be activated

during taurine transport due to its electrogenic nature

(Ramamoorthy et al. 1993). Electrogenic transport of

GABA and glutamate induces sufficient membrane de-

polarization for LVACC activation (Haugh-Scheidt et al.1995; Villalobos & Garcia-Sancho, 1995) and a direct

interaction of intracellular taurine with LVACC is also

possible (del Olmo et al. 2000).

We conclude now that taurine accumulation triggering

long-term enhancement of synaptic responses in the two

studied brain structures is provided by different transport

systems: striatal LLETAU needs activation of the taurine

(not GABA) transporter while GABA (not taurine) trans-

porters are involved in hippocampal LLETAU. We cannot

exclude, however, additional routes of taurine entry in the

striatum, since LLETAU was still present in some striatal

slices from TAUT KO mice.

Is LLETAU a physiological phenomenon? Although this

question cannot be answered directly at present, increased

accumulation of taurine in striatal neurones (Hilgier et al.1999) in asymptomatic stages of hepatic encephalopathy

may be related to LLETAU. The modulation of synaptic

transmission in pathological states requiring taurine’s role

as an osmolyte may contribute to the disturbances of fine

motor and cognitive functions found in hepatic enceph-

alopathy.

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Acknowledgements This work was supported by Deutsche Forschungsgemeinschaft,SFB 575, a Lise-Meitner-Stipendium to O.A.S, the FriedrichThyssen Foundation (D.H.) and the Russian Foundation for BasicScience, 01-04-48304 to A.N.C.

Synaptic potentiation by taurineJ Physiol 550.3 919