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. 2003 Jun 24;550(Pt 3):911–919. doi: 10.1113/jphysiol.2003.045864

Taurine-Induced Long-Lasting Enhancement of Synaptic Transmission in Mice: Role of Transporters

O A Sergeeva *, A N Chepkova †, N Doreulee *, K S Eriksson *, W Poelchen *, I Mönnighoff *, B Heller-Stilb *, U Warskulat ‡, D Häussinger ‡, H L Haas *
PMCID: PMC2343077  PMID: 12824447

Abstract

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.

Taurine, a sulfonated β-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 et al. 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 et al. 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 encephalopathy 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 ionotropic 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 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.

METHODS

Field potential recordings

Genotyped 6- to 11-week-old TAUT KO (n = 11) and wild-type (WT, n = 10) mice, the male and female offspring of the same heterozygous breeding pairs, and male C57BL/6 mice of the same age (n = 22) were used for electrophysiological experiments. All experiments were conducted in compliance with German Law and with the approval of Bezirksregierung Duesseldorf. The animals were decapitated and the brains rapidly removed, placed in ice-cold Krebs-Ringer solution and cut into horizontal slices, 400 μm thick, using a Vibroslicer (Campden Instruments). The slice preparations included the neostriatrum, the neocortex and the hippocampus (Fig. 1A). After 2–3 h preincubation at room temperature a single slice was transferred to a recording chamber and submerged in a continuously flowing (1.5 ml min−1) medium warmed to 32–33 °C. Both preincubation and perfusion media contained (mm) 124 NaCl, 3.7 KCl, 1.24 NaH2PO4, 1.3 MgSO4, 2.0 CaCl2, 26 NaHCO3, glucose 20 and were continuously saturated with a 95 % O2-5 % CO2 mixture.

Figure 1. Illustrations of a horizontal brain slice (A) and long-lasting enhancement of hippocampal synaptic transmission (B).

Figure 1

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The time course of CA1 field potential amplitudes (fEPSP) is illustrated in B. Taurine-induced long-lasting enhancement (LLETAU) of synaptic field responses in control (▪) is unchanged in the presence of the taurine uptake inhibitor guanidinoethyl sulphonate (GES, ▪). Times of taurine (TAU, 10 mm) and GES applications are indicated by horizontal bars. Abbreviations in A: CX, cortex; CS, corpus striatum; GP, globus pallidus; Th, thalamus; HPC, hippocampus.

Low-resistance glass micropipettes filled with perfusion medium were used to record field potentials in the neostriatum and in the stratum radiatum of the CA1 area of the hippocampus. A bipolar Ni-Cr stimulation electrode was placed on the white matter between the cortex and the neostriatum to stimulate the cortico-striatal axons, and on the hippocampal radial layer to stimulate Schaffer collaterals. The positions of stimulating and recording electrodes are indicated on the schematic picture of a horizontal slice (Fig. 1A). Constant-voltage pulses of 80 μs duration were applied 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 control recording, 30 min perfusion with 10 mm taurine and a 60 min washout period. Taurine (Sigma) was dissolved in the perfusion medium containing 10 mm glucose instead of 20 mm glucose to maintain the constant osmolarity and was applied by switching the two-tap perfusion system to the reservoir with taurine-containing medium. The osmolarity of solutions was controlled with an osmometer (Knauer, Berlin, Germany). In control experiments, glucose (10 mm) substitution with sucrose did not affect field potentials. Nipecotic acid was obtained from Sigma and guanidinoethyl sulphonate was from Toronto Research Chemicals.

Fifteen consecutive responses (5 min recording) were averaged off-line to generate one data point. The characteristic field potential evoked by cortical white matter stimulation consisted of two negative potentials: the first (N1) reflecting a fibre potential and 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 field EPSP slope were measured in the striatum and the hippocampus, respectively. All values were normalized to the mean value over the 30 min control period. The data were expressed as means ± s.e.m. and analysed statistically using Student's t test (two-tailed) and Fisher's exact probability test.

Real-time RT-PCR

Total cellular mRNA was isolated from the hippocampus and the dorsal striatum of either side using a mRNA isolation kit (Pharmacia Biotech) according to the manufacturer's protocol. Total mRNA was eluted from the matrix with 200 μl of RNase-free water. For reverse transcription, 8 μl of eluted mRNA was added to 7 μm reagent mixture prepared according to the protocol of the ‘first strand cDNA synthesis kit’ (Pharmacia Biotech). After incubation for 1 h at 37 °C the reverse transcription reaction was stopped by freezing at −20 °C. The reverse-transcription reactions were not normalized to contain the equivalent amounts of total mRNA. The PCR was performed in a PE Biosystems GeneAmp 5700 sequence detection system using the SYBR green master mix kit. Each reaction contained 2.5 μl of the 10xSYBR green buffer, 200 nm each of dATP, dGTP and dCTP, 400 nm dUTP, 2 mm MgCl2, 0.25 units of uracil N‘-glycosylase, 0.625 units of Amplitaq Gold DNA polymerase, 10 pM forward and reverse primers, 5 μl of 1:4 diluted cDNA, and water to 25 μl. Primers used for the semiquantitative analysis were as follows: β-actin forward: 5′-CGT GAA AAG ATG ACC CAG ATC ATG TT-3′; β-actin reverse: 5′-GCT CAT TGC CGA TAG TGA TGA CCT G-3′; TAUT forward: 5′-GAA AGA CTT CCA CAA AGA CAT CC-3′; TAUT reverse: 5′-GTA CTG GCC TAT GAT GAC CTC C-3′. The reactions were performed in MicroAmp 96-well plates or in optical tubes capped with MicroAmp optical caps. The reactions were 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‘-glycosylase and activate the Amplitaq Gold polymerase, followed by 40 cycles of 15 s at 95 °C, 1 min at 60 °C. The PCR reactions were subjected to a heat dissociation protocol (PE Biosystems 5700 software). Following the final cycle of the PCR, the reactions were heat denaturated over a 35 °C temperature gradient at 0.03 °C s−1 from 60 to 95 °C. Each PCR product showed a single peak in the denaturation curves. The identity of PCR products with the known cDNA sequences was determined as described before (Sergeeva & Haas, 2001). Standard curves for real-time PCR protocols with both primer pairs obtained with sequential dilutions of one cDNA sample (down to 1:1000) were found to be optimal (linear regression coefficients were 0.99 and 0.97 for the β-actin and TAUT respectively, P < 0.01).

The real-time PCR data were plotted as the fluorescence signal versus the cycle number. The PE Biosystems 5700 sequence detection system software calculates the ΔRn (Reporter, normalized) using the equation:

graphic file with name tjp0550-0911-mu1.jpg

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 midpoint of logΔRnversus cycle number plot. The Ct value is defined as the cycle number at which the ΔRn crosses this threshold. The change in taurine transporter (TAUT) cDNA (target gene) relative to the β-actin endogenous control was determined by:

graphic file with name tjp0550-0911-mu2.jpg

where

graphic file with name tjp0550-0911-mu3.jpg

Time x is any time point and time 0 represents the 1 × expression of each gene under conditions of serum starvation. Relative quantification of gene expression using the 2-ΔΔCt method correlated with the absolute gene quantification obtained in standard curves (Winer et al. 1999).

Immunohistochemistry

Horizontal slices of 0.5–1 mm thickness containing the striatum and the hippocampus were fixed for 8 h in phosphate-buffered 4 % paraformaldehyde (pH 7.4) at 4 °C, cryoprotected in sucrose, cryosectioned at 24 μm thickness, and mounted on gelatin-coated slides. All antibody incubations and washes were carried out in phosphate-buffered saline with 0.25 % Triton X-100 (pH 7.4) and all antibody solutions contained 2 % normal swine serum. To determine the localization of the taurine transporter we used a rabbit anti-taurine transporter serum (Tau11-A, DPC Biermann, Bad Nauheim, Germany) diluted 1:500-1:1000. Primary antiserum was applied to the sections for 24 h at 4 °C, and the following steps were performed at room temperature. After the incubation with primary antibody, the slides were incubated with a 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) for 2 h. The immunoreactivity was then visualized by an 8–12 min incubation in a solution of 0.03 % 3,3′-diaminobenzidine tetrahydrochloride, 0.015 % H2O2, and 0.1–0.2 % NiCl2 in Tris-HCl (pH 7.6), which yielded a black reaction product. When the primary antiserum was replaced with normal rabbit serum, the staining was absent.

Determination of taurine levels

Different brain structures were dissected under a microscope and the freshly removed tissues were frozen in liquid nitrogen and then stored at −70 °C. Frozen tissue was homogenized and weighed five times without thawing. Proteins were removed by incubation in 10 % sulfosalicylic acid on ice for 1 h. After centrifugation for 10 min at 21 000 g, the lipids were extracted from the supernatant with dichloromethane. Plasma was added to an equal amount of 10 % sulfosalicylic acid. Taurine content was measured in a BioChrom20 amino acid analyser (Amersham-Pharmacia Biotech, Freiburg, Germany).

RESULTS

Taurine transport and LLETAU

Bath application of taurine (10 mm for 30 min) to hippocampal 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 hippocampus (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 hippocampal and cortico-striatal slices, they developed a long-lasting enhancement, LLETAU. The data on taurine-induced enhancement of the CA1 field EPSP and cortico-striatal postsynaptic responses are summarized in Table 1 and illustrated in Figs 1, 2 and 3.

Figure 2. Taurine evokes long-lasting enhancement of synaptic transmission (LLETAU) in hippocampal slices from both wild-type (WT) and TAUT knockout (KO) mice.

Figure 2

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A 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 18 TAUT KO mice. Taurine (TAU, 10 mm) was applied for the time periods indicated by horizontal bars. C, representative field potentials in WT mouse. Traces are 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 from hippocampal slices of 10 WT and 8 TAUT KO mice. Data points in each experiment were normalized on the maximal response amplitude.

Figure 3. LLETAU of synaptic transmission in striatal slices from WT but not from TAUT KO mice.

Figure 3

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A and B, time courses of changes in the second negative component of the field (N2). Diagrams summarize time courses of postsynaptic changes in WT (n = 18) and TAUT KO (n = 20) mice. Taurine (10 mm) was applied for the time periods indicated by the horizontal bars. C, representative field potentials in WT mice are inserted; traces are 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 from striatal slices of 8 WT and 10 TAUT KO animals. Data points in each experiment were normalized to the maximal response amplitude.

Table 1.

Long-lasting enhancement of synaptic transmission by taurine (LLETAU) in the striatum and the hippocampus

Striatum Hippocampus
Mice n slces total LLETAU magnitude (occurrence) n slices total LLETAU magnitude (occurrence)
TAUT WT 18 160.8 ± 8.0%, n = 17 16 138.9 ± 5.8%, n = 14
(94%) (88%)
TAUT KO 20 137.4 ± 6.6%, n = 8 18 130.2 ± 5.4%, n = 14
(40%)*** (78%)
C57BL/6 18 146.2 ± 5.5%, n = 17 15 144.5 ± 7.6%, n = 14
(94%) (93%)
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WT, wild-type; KO, TAUT −/−.

***

P < 0.001.

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 (Fig. 2 and Fig. 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 t test) 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 versus 59.0 ± 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).

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 Fig. 2 and Fig. 3.

GABA transporter antagonist prevents LLETAU in hippocampus but not in striatum

Under 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).

Figure 4. The GABA transporter antagonist nipecotic acid (NPA) blocks LLETAU in hippocampus, but not in striatum.

Figure 4

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Time courses of changes in the second negative component of the field potential in striatum and the field EPSP (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 field potentials from striatum of C57BL/6 are inserted at the right.

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 potentiation of field responses (Fig. 4). LLETAU in the control slices peaked at 20–30 min after taurine withdrawal 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 hippocampus (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 the striatum

Semiquantitative real-time PCR revealed TAUT mRNA levels to be 2.5 times higher in the dorsal striatum than in the hippocampus. The values, 2-ΔΔCt, 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 the neuropil, possibly of dendrites, no somatic staining could be clearly discerned in either structure, even under higher magnification.

Figure 5. Immunostaining for the TAUT protein.

Figure 5

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a, the strongest staining is seen in the neuropil of the striatum and in a narrow band in the hippocampal CA3 region. Scale bar, 1 mm. Higher magnifications: hippocampal CA3 (b) and dorsal neostriatal (c) fields. No stained somata can be identified with certainty. Scale bars, 50 μm.

Taurine content in discrete brain areas

Taurine 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.

Table 2.

Concentration of taurine in microdissected brain areas from WT, heterozygote (HT) and TAUT KO mice

Taurine (μMol (g wet weight)−1)
Brain area WT, n = 4 HT, n = 4 KO, n = 4
Brain stem 4.03 ± 0.32 2.31 ± 0.21** 0.39 ± 0.02***
Cerebellum 8.77 ± 0.72 5.40 ± 0.64* 0.26 ± 0.07***
Dorsal striatum 9.02 ± 0.86 7.23 ± 0.54 n.s. 0.41 ± 0.15***
Hippocampus 8.66 ± 0.27 4.79 ± 0.24*** 0.18 ± 0.01***
Cerebral cortex 8.51 ± 0.64 4.81 ± 0.57** 0.20 ± 0.02***
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***

P < 0.001

**

P < 0.05

*

P < 0.05, n.s. not significant; comparison of HT or KO with WT, same brain region.

DISCUSSION

Our 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 et al. 2002).

In the CA1 area of the hippocampus, where the involvement 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 mechanisms 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 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 heterozygous 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 hybridization 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 augmentation 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 transporters, 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 hippocampus (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 depolarization 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) transporters 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 encephalopathy.

Acknowledgments

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

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