Theanine, γ-glutamylethylamide, a unique amino acid in tea leaves, modulates neurotransmitter concentrations in the brain striatum interstitium in conscious rats
- 2.5k Downloads
Theanine (γ-glutamylethylamide) is one of the major amino acid components in green tea and can pass through the blood-brain barrier. Recent studies suggest that theanine affects the mammalian central nervous system; however, the detailed mechanism remains unclear. In this study, we demonstrated the effect of theanine on neurotransmission in the brain striatum by in vivo brain microdialysis. Theanine injection into the rat brain striatum did not increase the concentration of excitatory neurotransmitters in the perfusate. On the other hand, theanine injection increased the concentration of glycine in the perfusate. Because it has been reported that theanine promotes dopamine release in the rat striatum, we investigated the glycine and dopamine concentrations in the perfusate. Co-injection of glycine receptor antagonist, strychnine, reduced theanine-induced changes in dopamine. Moreover, AMPA receptor antagonist, which regulates glycine and GABA release from glia cells, inhibited these effects of theanine and this result was in agreement with the known inhibitory effect of theanine at AMPA receptors.
Keywordsl-theanine Dopamine Neurotransmission Glycine AMPA receptors Microdialysis
In this study, we demonstrated the effect of theanine on inhibitatory neurotransmission and other neurotransmitters in the central nervous system by in vivo brain microdialysis. In addition, we tried to explain the action mechanism of theanine on brain neurotransmission involving dopamine neurotransmission in the brain striatum.
Materials and methods
Male Wistar rats (270 g weight; SLC, Hamamatsu, Japan) were kept in individual wire cages in a temperature- and humidity-controlled room (24°C and 55% relative humidity) under regular lightning conditions (12 h light: dark cycle), and given food and water ad libitum. This experiment was carried out in accordance with Guidelines for the Care and Use of Laboratory Animals of the University of Shizuoka that refer to the American Association for Laboratory Animals Science. Eight animals were used in all experiments.
Measurement of neurotransmitter concentration by brain microdialysis
The guide cannula of the microdialysis probe in the striatum was stereotaxically implanted 0.2 mm posterior to the bregma, 3.0 mm lateral to the midline, and 3.5 mm ventral to the cortical surface according to the atlas of Paxinos and Watson (Paxinos and Watson 1986). One day after the operation, the dummy cannula was removed from the guide cannula, and the dialysis probe was inserted. This dialysis probe (MI-A-I-8-03, Eicom, Japan) had a dialysis membrane (at molecular weight cut-off of 5,000 and 3 mm protruding from the guide cannula) and a side tube to microinject theanine. The probe was perfused with Ringer’s solution (147 mM Na+, 4 mM K+, 2.3 mM Ca2+, and 155.6 mM Cl-, pH 6.0) at a rate of 2 μl/min (ESP-64; Eicom, Japan). l-theanine (0.2 μmol/2 μl perfusate/min) was injected for 1 h and Ringer’s solution was re-perfused. In antagonist co-injection cases, each antagonist was injected with perfusate (2 μl perfusate/min) for 1 h before theanine injection and switched both theanine and each antagonist containing perfusate (2 μl perfusate/min). Theanine and antagonists were infiltrated into the tissue via dialysis membranes and the permeability of the membranes was approximately 10%.
The perfusate from the striatum (40 μl) for measuring dopamine was collected sequentially and was injected into the HPLC every 20 min via an automatic injector (AS-10; Eicom, Japan). Dopamine was separated by a reverse-phase HPLC using an MA-ODS column (5 μm particles, 3.0 × 150 mm; Eicom, Japan) maintained at 25°C by a column oven (ATC-300; Eicom, Japan), and was detected with an electrochemical detector (CB-100; Eicom, Japan). The mobile phase was composed of 0.1 M citric acid buffer, pH 3.9, containing 12% methanol, 20 mM EDTA and 160 mg/l sodium-1-octane-sulfonate. The graphite electrode (WE-3G; Eicom, Japan) was set at +0.65 V (vs. Ag/AgCl). The perfusates for measuring amino acids were collected into collecting tubes automatically every 30 min via the microsampler (Univentor, Malta) maintained at 4°C. The collected perfusates (60 μl) were diluted twice with 0.1N HCl. Ten microliters of the diluted perfusates was mixed with 5 μl o-phthaldialdehyde and 5 μl of 2 mercaptoethanol (OPA Reagent Set; Wako, Japan), and reacted for precisely 3 min. Twenty microliters of the reacted perfusates was injected into the chromatography system. The analytical ODC column (2 μm particles, 4.6 × 100 mm; MERCK, Germany) was maintained at 40°C and the detection wavelength was Ex: 340, Em: 420 (HP1049; Agilent, USA). The mobile phase used for HPLC analysis was composed of two eluants. Eluant A was 0.1 M phosphate buffer containing 10% ethanol, pH 6.0; eluant B was 100% ethanol: elution gradients were: initial, 86% A, 14% B; 0–16 min, and changed to 29% A, 71% B; 16–25 min, maintained for 25–32 min to wash the column, then re-equilibration with 86% A, 14% B for 15 min prior to the next step. The flow rate was kept constant at 1.0 ml/min. After completion of the experiment, the position of the microdialysis probe was histologically examined.
Data presentation and statistical analysis
The concentrations of dopamine and amino acids in the perfusate were expressed as percent change from the averaged basal values. In the measurement of dopamine concentration, basal values were averaged for four samples of perfusate before the theanine injection (pg dopamine/40 μl perfusate/20 min). In the measurement of amino acid concentration, basal values were averaged for three samples of perfusate before the theanine injection (glutamic acid, aspartic acid, glycine, glutamine, asparagine respectively; pg amino acid/60 μl perfusate/30 min). Differences between basal values and each fraction value of perfusates were analyzed using the Tukey-Kramer test. Differences for each reagent were analyzed using Student’s t-test. In all cases, P < 0.05 was considered significant. Results are expressed as the mean ±SEM.
l-theanine was obtained from Taiyo Kagaku Co., Ltd. (Yokkaichi, Japan). NBQX disodium salt (NBQX), strychnine and 5,7-dichlorokynurenic acid (5,7-DCKA) were purchased from Sigma Chemical Company (St. Louis, MO, USA).
Basal levels of glutamic acid, aspartic acid and glycine concentrations in the perfusate were Glu: 20.8 ± 3.3, Asp: 3.86 ± 0.3 and Gly: 23.2 ± 3.1 ng/60 μl perfusate/30 min respectively, before theanine injection. Injection of theanine did not significantly change glutamic acid concentration in the perfusate (Fig. 2b, c). Aspartic acid concentration was reduced approximately 50% from the basal level. Glycine concentration increased about sixfold compared with the basal level. The changes in these amino acid concentrations tended to return to the basal value following re-perfusion of Ringer’s solution.
Dopamine is related to emotion, behavior and brain disorders such as Parkinson’s disease (Bosboom et al. 2003; Tessitore et al. 2002). In vivo microdialysis studies suggested that glutamic acid and dopamine were released simultaneously during ischemia (Globus et al. 1988), while others have demonstrated that glutamic acid was required to enhance the extracellular concentration of dopamine in vivo (Moghaddam et al. 1990). In addition, some studies suggested that dopamine could modify glutamate neurotransmission. (Moghaddam et al. 1990; Yadid et al. 1993; Koga and Momiyama 2000). Because the chemical structure of theanine is similar to glutamic acid, some studies of theanine examined its relationship to glutamic acid function and metabolism (Sugiyama et al. 2003; Kakuda et al. 2002a; Tsuge et al. 2003; Oda et al. 1980); however, the glutamate receptor antagonist MK-801 did not affect the increase of dopamine concentration by theanine injection in the rat striatum (Yokogoshi et al. 1998a). This result suggested that theanine did not affect glutamate neurotransmission. On the other hand, some reports suggested that theanine influenced glutamate transporter systems (Sugiyama et al. 1998; Sugiyama et al. 2001; Sadzuka et al. 2001). In a previous study, we showed that injection of l-trans-2, 4-PDC, a glutamate transporter blocker, increased dopamine concentration dramatically and increased glutamic acid concentration in the interstitium. From these results, we expected that l -trans-2,4-PDC injection caused glutamic acid reuptake inhibition, increasing glutamic acid concentration in the interstitium; thus, glutamic acid might promote dopamine neurotransmission. It was observed that aspartic acid concentration was also increased by l-trans-2,4-PDC injection. Aspartic acid is known as an endogenous glutamate receptor agonist, which combines with the NMDA receptor (Olverman et al. 1988).
Theanine injection increased dopamine concentration up to 300% of the basal value in the interstitium and this result was similar to the value obtained in a previous study (Yokogoshi et al. 1998a). Theanine may influence interstitium dopamine concentration either directly or indirectly, e.g., via glutamatergic nerve terminals; however, theanine had no effect on interstitial glutamic acid concentration, and interstitial aspartate was even reduced by theanine. These results suggested that theanine did not increase excitatory neurotransmitter concentrations in the interstitium, such as l-trans-2,4-PDC, and the effect of theanine on dopamine concentration in the interstitium may be independent of the glutamic acid excitatory neurotransmission pathway.
Theanine injection increased glycine concentration dramatically in the interstitium. Glycine is known as a major inhibitory neurotransmitter in the spinal cord and brainstem, and is concerned with processing motor and sensory information, and disorders such as epilepsy. Recent studies showed that glycine was released from granule cells and astrocytes, and there are many glycine receptors in the central nervous system. In the central nervous system, glycine and these receptors mediate inhibitory neurotransmission in the same manner as GABA and work simultaneous with GABA (Legendre 2001; Lopez et al. 2001). Moreover, it was revealed that glycine injection increases dopamine concentration in the rat striatum interstitium (Yadid et al. 1993). Thus, we examined whether theanine increased interstitial glycine concentration and whether glycine neurotransmission contributed to the increase in interstitial dopamine.
Strychnine, a glycine receptor antagonist, inhibited the increase of dopamine concentration by theanine injection in the interstitium. This result suggested that the increases of glycine in the interstitium by theanine might have influenced dopamine release via glycine receptors. Although glycine is known as a major inhibitory neurotransmitter, glycine combines with the glycine binding site of the NMDA receptor and enhances excitatory neurotransmission (Xu et al. 1999; Kuhse et al. 1995; Nong et al. 2003). Glycine enhanced NMDA evoked dopamine release, and 7-chloro-kynurenate, an NMDA antagonist, acting on a glycine site, markedly reduced this response (Martinez et al. 1992); however, 5,7-DCKA injection did not prevent the increase of dopamine concentration by theanine injection in this study. These results suggested that theanine-increased glycine influenced interstitial dopamine via glycine receptors, and not NMDA receptors.
In this study, dopamine concentration data have negligible error bars and glycine data have large error bars. From these data, we expected that not only glycine but also other factors were involved in the effect of theanine on dopamine concentration in the perfusate. It was shown that glycine is co-localized with GABA in astrocytes and interneurons (Ross and Soltesz 2001; Bureau and Mulle 1998; Xu et al. 1999; Levi and Patrizio 1992), and glycine and GABA are released simultaneously and work synergistically on different targets (Bohlhalter et al. 1994). We expected that the GABAergic nervous system may also be influenced by theanine injection and is involved in dopamine concentration increase in the perfusate; however we did not examined the effect of theanine on intersutitial GABA concentration and GABA receptors in this study.
It was reported that inhibitory neurotransmitters, such as glycine and GABA, are released by activating α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, ionotropic glutamate receptors (Ross and Soltesz 2001; Bureau and Mulle 1998; Xu et al. 1999), and recent reports showed that activating AMPA/kainate receptors facilitated electrically evoked dopamine efflux in guinea pig striatal slices, and glycine and GABA release from astrocytes and interneurons (Antonelli et al. 1997; Han et al. 2000). In addition, it was shown that theanine bound to AMPA receptors not to NMDA receptors (Kakuda et al. 2002a). Thus, we supposed AMPA receptors to be involved in the effects of theanine on glycine levels in the perfusate. NBQX, an AMPA/kainate receptor antagonist, prevented both theanine-induced increases in perfusate dopamine and glycine levels. This result suggested that AMPA/kainate receptors might be involved in the early phase of theanine-evoked neurotransmitter changes in the perfusate. Moreover, because theanine did not change the glutamate level in the perfusate (Fig. 3a), theanine itself might have acted on AMPA/kainate receptors directly (Kakuda et al. 2002a).
This work was supported in part by grants for scientific research from Shizuoka Prefecture, and the 21st Century COE program from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
- Kimura R, Murata T (1971) Influence of alkylamides of glutamic acid and related compounds on the central nervous system. I. central depressant effect of theanine. Chem Pharm Bull (Tokyo) 19:1357–1361Google Scholar
- Kimura R, Murata T (1986) Effent of theanine on norepinephrine and serotonin levels in rat brain. Chem Pharm Bull (Tokyo) 34:3053–3057Google Scholar
- Oda Y, Taguchi H, Masaoka N, Minami K, Honda S, Okada K (1980) Synthesis and antibacterial activities of theanine-containing oligopeptides. Chem Pharm Bull (Tokyo) 28:3549–3554Google Scholar
- Paxinos G, Watoson C (1986) The rat brain in stereotaxic coordinates. Academic Press, New YorkGoogle Scholar