Skip to main content
NCBI home page
IBRO Neuroscience Reports logoLink to IBRO Neuroscience Reports
. 2025 Jun 6;19:63–71. doi: 10.1016/j.ibneur.2025.05.015

Effect of L-theanine on cerebellar granule cell migration related to cognitive disorders

Mai Ibrahim a, Matsuda Tomoko b, Kuriya Kenji a, Ozeki Makoto c, Abe Aya c, Hayato Umekawa a,d, Masahiro Nishio a,⁎
PMCID: PMC12181011  PMID: 40547867

Abstract

Introduction

Cerebellar granule cell migration plays a crucial role in cerebellum development, and any abnormalities in CGC migration can lead to significant neurological disorders such as anxiety, a common psychological disorder that impacts a person's emotional, physical, and social health. L-theanine, an amino acid found in green tea, demonstrates neuroprotective properties and regulates the release of neurotransmitters by stimulating CGC migration. This study investigated the impact of L-theanine on CGC migration related to cognitive disorders.

Methods

ddY male mice treated with a single oral dose of L-theanine at varying concentrations (10 mg/kg) were assessed for anxiety, learning, and memory using the maze test and the Morris Water Maze test, where the average completion time and escape time of the mice were considered indicators of cognitive performance. CGC microexplants were isolated from newly born C57BL/N6 mice and treated with a series of increasing concentrations of L-theanine. The migration distance of the CGC under the different L-theanine concentrations was assessed after 24, 48, and 72 h post-treatment using phase-contrast microscopy and image analysis software.

Results and conclusion

Mice's anxiety symptoms improved based on their performance on the maze test after treatment with L-theanine at 5 mg/ml. However, L-theanine at 0.05 mg/ml enhanced learning and memory abilities. Compared to other concentrations, L-theanine at 1 µM yielded the longest migration distance for CGC in vitro. Therefore, L-Theanine may serve as a potential therapeutic agent in supporting cerebellar development and enhancing cognitive skills. Further investigation is required to fully elucidate the molecular mechanisms and therapeutic potential of L-theanine in neurodevelopmental disorders.

Keywords: L-theanine, Maze, Anxiety, Cerebellar granule cell, Cell migration, Microexplant

Highlights

  • •

    L-theanine can improve the cognitive performance of mice at the maze test and Morris Water Maze test.

  • •

    A low concentration of L-theanine (1 µM) positively supports CGC migration.

  • •

    L-theanine has a potential role in brain development.

1. Introduction

The central nervous system (CNS) development follows a common pattern of localized proliferation, followed by cell migration and differentiation (Borghesani et al., 2002). The cerebellum encloses more than half of the brain's neurons and is responsible for many cognitive abilities. The adult cerebellar cortex consists of three layers, the granular layer, the Purkinje cell layer, and the molecular layer (Paredes et al., 2016). The granular layer, which is the deepest layer; is packed with cerebellar granule cells (CGCs), which are essential for information transmission to Purkinje cells. CGC progenitors move from the rhombic lip during the early stages of postnatal development to form the external granule cell layer (EGL), where they multiply, differentiate, and continue to migrate via the molecular layer (ML) until they reach the internal granule cell layer (IGL) (Borghesani et al., 2002; Butts et al., 2014). Purkinje cells play a critical role in the survival and growth of CGCs. Chronic depolarization is necessary for these cells to avoid apoptosis in vitro. This depolarization is accomplished by activating N-methyl-D-aspartate (NMDA) receptors, which raise intracellular calcium levels and support neuronal survival (Krämer and Minichiello, 2010).

L-theanine (γ-glutamylethylamide) is a non-protein amino acid predominantly found in green tea (Camellia sinensis), constituting approximately 1–2 % of the dry weight of tea leaves and about 50 % of the total free amino acids. Clinically, L-theanine has been shown to reduce stress, improve immunity, promote weight loss, and protect cardiovascular health (Vuong et al., 2011). L-theanine also has neuroprotective properties that are associated with its structural resemblance to glutamate, causing it to act as an antagonist (Bryan, 2008). Glutamate is the brain's primary excitatory neurotransmitter; it can interact with glutamate receptors like N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methoxazole-4-propionic acid (AMPA), and kainate. L-theanine’s antagonistic action might be involved in dopamine and serotonin secretion in particular brain regions as well as neuroprotection against glutamate toxicity (Dramard et al., 2018). Animal studies suggest that L-theanine, which acts as a low-affinity glutamate receptor antagonist, binds to NMDA receptors and protects neurons from excitotoxic damage (Kakuda et al., 2002, Nathan et al., 2006). In animal experiments, L-theanine has shown antidepressant and anti-stress properties (Hidese et al., 2019). L-theanine exerts its effects through many mechanisms, including preventing glutamate absorption, increasing gamma-aminobutyric acid (GABA) levels, promoting dopamine release, and modifying serotonin levels (Wang et al., 2022). Research suggests that the stress-reducing properties of L-theanine could be directly translatable to its effects on learning and memory.

Glutamate has a role in a variety of behavioural and physiological processes, including synaptic plasticity, memory, learning, and cellular metabolism. Because L-theanine is structurally similar to glutamate, it's vital to think about how it might interact with glutamate receptors. Since it can mimic and regulate glutamate, which is important in synaptic plasticity, learning, and memory, L-theanine can help with learning and memory disorders (Robbins and Murphy, 2006, Williams et al., 2020).

After consumption, it reaches peak plasma concentration after 45–50 min, and it is completely removed in vivo in 24 h (Li et al., 2022). It has been proved that L-theanine increases the number of immature neurons and progenitor cells in the dentate gyrus, which indicates that it may promote neurogenesis in the growing hippocampus (Takeda et al., 2011).

The connection between anxiety and cerebellar granule cells is an emerging field of study, emphasizing the cerebellum's function beyond motor control to encompass emotional regulation. According to Gilbert (2014), anxiety is a common mental illness that affects one's social, physical, and mental well-being. It is a behavioural condition that is frequently linked to abnormalities in the cerebellum. Since they give Purkinje cells sensory, proprioceptive, and contextual information to alter behaviour in various settings, CGCs are crucial for cerebellar function (Jiang et al., 2023).

L-theanine has been shown to increase calcium concentration within cultured cerebellar granule neurons when administered in the micromolar range (Sebih et al., 2017). Both clinical and experimental evidence strongly suggest a functional role for the cerebellum in anxiety and anxiety behaviours, where the granule cells might be involved. L-theanine has been shown to exert anxiolytic effects, potentially influencing cerebellar activity and granule cell function, which may play a role in anxiety regulation. (Jiang et al., 2023).

Here, through this study, we investigated the impact of different L-theanine concentrations on cellular migration in vitro and on animal behaviour focusing on the lowest effective doses of L-theanine, which sheds additional light on how well it can enhance cognitive performance. We used lower concentrations compared with the previous study to estimate the least amount of L-theanine that can give an action in vivo.

2. Methods

2.1. Maze test

The maze is customized in Taiyo Kagaku Co. Ltd., Yokkaichi, Japan, with a special design to induce anxiety levels (130 cm × 140 cm and height 25 cm). (Fig. 1)

Fig. 1.

Fig. 1

Open in a new tab

Maze is customized in Taiyo Kagaku Co. Ltd., Yokkaichi, Japan, with a special design to induce anxiety levels. Maze’s dimensions are 140 cm × 130 cm and 25 cm in height, made from wood. As mice were placed at the starting point (S) and videotaped, their movement was recorded until goal point (G).

L-theanine was used at 3 different concentrations to measure the behavioral effects of L-theanine. 18 male ddY mice (11 weeks old) (SLC, Japan) were divided into 4 groups: a control group and 3 groups each treated with a different dose of L-theanine: low, medium, and high doses of L-theanine. To initiate the test session, the mice were placed individually at the start point of the maze just 30 min after L-theanine administration, and the average time from the starting point (S) to the goal point (G) was recorded.

L-Theanine (Taiyo Kagaku Co. Ltd., Yokkaichi, Japan) was used at high (5 mg/ml), medium (0.5 mg/ml), and low (0.05 mg/ml) concentrations. The consumed L-theanine solution was prepared freshly just before the experiment. The body weight of each mouse in 4 groups at the beginning of the experiment was measured; hence, the dose of the L-theanine solution was calculated as 10 mg/kg.

2.2. Morris water maze (MWM)

Animals were tested using a spatial version of the Morris Water Maze (MWM) test (Pawlak et al., 2003, Morris et al., 1982) for five consecutive days. The water maze consisted of a circular pool filled with water at room temperature (diameter: 120 cm; height: 25 cm; water temperature: 24 ± 1°C). A platform (diameter: 10 cm) was hidden 1.5 cm below the surface of the water. The pool was virtually divided into four quadrants. The escape platform was kept at a fixed position in the center of one of the quadrants. The mice underwent five consecutive daily training trials over these five days, with each trial lasting until the mouse either found the platform or for a maximum of 60 s. All mice were allowed to rest on the platform for 15 s after each trial. A probe trial was conducted (Tuzcu and Baydas, 2006, Kuhad and Chopra, 2008, Kuhad and Chopra, 2007) on the sixth day to assess the extent of memory consolidation. During the probe trial, the mice were placed and released opposite the location where the platform had been. The single-probe trial consisted of a 30-second free swim in the pool without the platform. The amount of time spent in the target quadrant indicated the degree of memory consolidation that occurred after learning, and the percentage of time spent in the former platform location was recorded for the probe trial.

We tested 20 male ddY mice (6 weeks old) (SLC, Japan) on the testing day. We tested 3 different concentrations of L-theanine, as mice were given with L-theanine (5 mg/ml, 0.5 mg/ml, and 0.05 mg/ml). The water level in the pool increased to 1 in. higher than the platform. All mice were orally administered according to their body weight (10 ml/kg). (Fig. 2)

Fig. 2.

Fig. 2

Open in a new tab

Diagram representation of the spatial version of the Morris Water Maze (MWM) test. Mice were tested for seven consecutive days to assess spatial learning and memory.

2.3. Cerebellar granule cell microexplant

The C57BL/6N newborn mice were bred under supervision. All animal procedures were conducted at the Mie University Experimental Animal Facilities (2022–5) under a 12-h light/dark cycle. Food and water were available ad libitum. All experimental procedures adhered to the Mie University guidelines for laboratory animals, approved by the Institutional Animal Care and Use Committee of Mie University, and complied with the governmental guidelines (the Japanese Association of Laboratory Animal Facilities of National University Corporations).

Microexplants were prepared from freshly isolated cerebella of P3–P4 mice (3–4 cerebella used for each experiment) as previously described (Kokubo et al., 2009). Briefly, the meninges and choroid plexus were promptly liberated from the cerebellum, allowing it to exit the skull after the mice were decapitated. Subsequently, white matter and deep cerebellar nuclei were excised from the cerebellar slices using a surgical blade. Rectangular sections measuring 50–100 µm were then prepared from the residual tissue, which was primarily composed of the cerebellar gray matter. The extracted microexplants were cultured on 24-well plates coated with 100 μg/ml poly-D-lysine hydrobromide (Nacalai Tesque Co., Ltd., Kyoto, Japan) and 50 μg/ml laminin (FujiFilm Wako Pure Chemical Co., Ltd.). All procedures were performed in three independent experiments conducted in triplicate. The explants were incubated with Basal Medium Eagle with Earle’s salts (BME, Invitrogen) supplemented with 1 mg/ml Bovine Serum Albumin (BSA), 10 μg/ml insulin, 35 nM selenate, 5.5 µg/ml transferrin, 0.1 nM T4, 0.25 % glucose, 1 µg/ml aprotinin, 2 mM glutamine, N2 supplement, and penicillin/streptomycin.

2.4. Migration assay

Different concentrations of L-theanine (γ-glutamyl ethyl amide) (0.1 nM, 1 nM, 10 nM, 100 nM, 1 µM, 10 µM, 100 µM, and 1 mM, Taiyo Kagaku Co., Ltd., Japan) were introduced to freshly isolated cerebellar explants and incubated for 3 continuous days at 37°C and 5 % CO2 after a single addition at seeding. Control explants were incubated with the medium (BME) alone. Migration distances were measured from the edges of the microexplanted tissue to the cell body using a digital microscope and the Olympus cellSens standard software. The cell movement was monitored over three days, with images captured at 24, 48, and 72 h. Cell morphology was assessed using phase-contrast microscopy at 4x and 10x magnification. Migrating cerebellar granule cells were identified by their elongated, bipolar shape and directional alignment radiating from the explant core, consistent with previously described migratory profiles (Komuro and Yacuboa, 2003a)

2.5. Time-lapse assay

The same procedures performed for the migration assay were repeated; however, the incubation with the different concentrations of L-theanine was initiated at different time points after isolating the microexplants and culture initiation. The addition of L-theanine was initiated after 3, 12, 24, and 48 h post-isolation. The concentrations of L-theanine used in this experiment were 1 µM, 10 µM, 100 µM, and 1 mM. The microexplants were photographed under a light microscope after 72 h of culturing in the well plates.

2.6. Statistical analysis

Data were analyzed using one-way ANOVA followed by Tukey’s test and Dunnett’s test for multiple comparisons. Statistical significance was set at p < 0.05, indicating meaningful differences between groups. The data are presented as mean ± standard deviation (SD). All statistical analyses were performed using Microsoft Excel, and supplementary analyses were conducted using BellCurve for Excel (version 4.08) (Social Survey Research Information Co., Ltd.)

3. Results

3.1. Effect of L-theanine on animal behaviour (maze test)

The maze test was used to investigate the effect of L-theanine on animal behaviour. The findings showed that L-theanine affected cognitive function based on maze completion time in a concentration-dependent manner. Compared to untreated controls, animals treated with the lowest concentration (0.05 mg/ml) showed low effects, suggesting that low levels of L-theanine do not appear to affect cognitive function after a single dose administration. However, mice administered the medium concentration (0.5 mg/ml) showed a clear increase in maze completion time (p < 0.05), indicating improved decision-making or more exploratory behaviour, which may indicate more cognitive engagement. The maze completion time considerably increased at the maximum dosage (5 mg/ml), suggesting a major impact on cognitive processes, maybe due to enhanced focus, less impulsivity, or less stress. (Fig. 3a)

Fig. 3.

Fig. 3

Open in a new tab

Average time in seconds taken by mice in the maze. a. Average completion time taken by mice after 30 min of oral administration of ultra-pure water (control group), L-Theanine alone (0.05 mg/ml), (0.5 mg/ml), and (5 mg/ml) (n = 18). b. Average time of staying during maze test in seconds. The data are representative of three independent experiments conducted in triplicate and are expressed as mean ± SD. Individual data points are overlaid on each bar to show variability across animals (n = 4 per group). Different letters represent significant differences between L-theanine vs. control at One-Way ANOVA *p < 0.05, **p < 0.001.

L-theanine also has a dose-dependent effect on the grooming of mice’s behaviour, as after calculating the time and number of stays for each concentration of L-theanine, the same effect of maze completion time is observed, with L-theanine at high concentration (5 mg/ml) at a single dose showing the longest time of staying. (Fig. 3b)

3.2. Effect of L-theanine on learning and memory (Morris Water Maze test)

To examine whether L-theanine could induce cognitive abilities, we tested learning and memory using the Morris water maze (MWM) test. The mean escape latency for the trained mice was decreased throughout the learning trials in all the groups, and from the third day onwards, there was a significant difference in escape latency. Treatment with L-theanine at the dose of 0.05 mg/ml decreased the escape latency as compared to the CNT mice (Fig. 4a). However, there was a difference in escape latency between the L-theanine (0.5 mg/ml, 5 mg/ml) groups and the control group, as mice administered these doses showed a longer time in MWM. In the probe trial of the MWM study at the sixth day, which assesses how well the animals have learned and consolidated the platform location during the five days of training, the animals showed a significant difference. The percentage of time spent in the target quadrant was low in the mice group, which received L-theanine (0.05 mg/ml) later the next day. However, other groups showed a high percentage of time that mice spent in the target quadrant.

Fig. 4.

Fig. 4

Open in a new tab

Effect of L-theanine on learning and memory abilities using Morris Water Maze. a. Comparison of escape latency of mice during pre-test (average training trials) and L-theanine (0.05, 0.5, 5 mg/ml) and CNT (water only). b. Comparison of travel distance between pre-test (average training trials) and L-theanine (0.05, 0.5, 5 mg/ml) and CNT. (n = 20). The data are representative of three independent experiments conducted in triplicate and are expressed as mean ± SD. Individual animal values are plotted over each bar (n = 5 per group). Different letters represent significant differences between L-theanine vs. control at One-Way ANOVA *p < 0.05, **p < 0.001.

Administration of L-theanine to animals at low concentrations (0.05 mg/ml) resulted in a reduction in the time required for these animals to find the hidden target compared to the control group. However, increasing the concentration of the administered L-theanine resulted in an increase in the time needed by the moderate (0.5 mg/ml) and high (5 mg/ml) L-theanine concentration groups compared to the control. (Fig. 4b) One can observe a dose-dependent increase in the escape time by increasing the concentration.

3.3. Effect of different concentrations of L-theanine on CGC migration

The microexplants incubated with different L-theanine concentrations (ranging from 0.1 nM to 1 mM) exhibited varying migratory responses. In the microexplant cultures of the P3–P4 mouse cerebella, the granule cells migrated extensively away from the tissue center. L-theanine had a concentration-dependent influence on CGC migration, peaking at 1 µM. This suggests that L-theanine had a significantly beneficial effect on migration distance when compared to the control (p < 0.001). Migration distance was significantly reduced in a concentration-dependent manner when the concentration was raised above 1 µM. (Fig. 5).

Fig. 5.

Fig. 5

Open in a new tab

Effect of L-theanine on the migration distance of CGCs. a. Digital microscope images of CGCs treated with L-theanine at 0.1 nM, 1 nM, 10 nM, 100 nM, 1 µM, 100 µM, 10 µM, and 1 mM were taken 24, 48, and 72 h after treatment with L-theanine at 4x magnification. b, c, d. Quantitative analysis of the average migration distance for each concentration at 24, 48, 72 h respectively. The migration distance was measured in micrometers (µm) from the micro-explanted tissue edge to the migrated CGC body e. Color-enhanced images show cerebellar tissue microexplants and the radial migration of granule cells at 2nd day of L-theanine 1 µM. Insets (higher magnification 10x) highlight the typical bipolar morphology of migrating CGCs, with a leading process oriented away from the explant core and a trailing process behind the soma. Sample size (n) is indicated above each bar, representing the number of independent migrated CGC per condition. The data are representative of three independent experiments conducted in triplicate and are expressed as mean ± SD. Different letters represent significant differences between L-theanine vs. control at One-Way ANOVA *p < 0.05, **p < 0.001.

In contrast to cells treated with lower doses, which showed reduced migration distances, cells treated with 1 µM moved substantially farther (p < 0.001). It should be mentioned that migration distances at concentrations below 1 µM differed significantly from the control. Conversely, concentrations exceeding 1 µM showed a concentration-dependent effect on migration distance (Fig. 5).

3.4. Time-lapse microscopy of CGC migration under different concentrations of L-theanine

The migration of CGCs incubated with different concentrations of L-theanine (1 µM, 10 µM, 100 µM, and 1 mM) was assessed 72 h after seeding to evaluate the effects of L-theanine on migration phases. Incubation with the different concentrations of L-theanine was initiated at different time points after isolating the microexplants and culture initiation. Consistent with previous experimental results, 1 µM of L-theanine exhibited initial improvement in the migration distance when added as early as 3 h after the culture initiation, indicating that the migration was activated early at this dosage. This effect was increased after 12 h and retained even after the treatment was initiated 24 h post-seeding, compared to the control and higher concentrations. Notably, adding L-theanine 48 h after seeding did not result in a significant effect on the migration across all concentrations compared to control-treated cells (Fig. 6).

Fig. 6.

Fig. 6

Open in a new tab

Effect of L-theanine at different starting time points on the CGC migration. Time-lapse analysis of the CGCs migration was compared after 72 h of starting the culture. L-Theanine was added to the explants at different concentrations at 3, 12, 24, and 48 h after starting the culture. Sample size (n) is indicated above each bar, representing the number of independent migrated CGC per condition. The data are representative of three independent experiments conducted in triplicate and are expressed as mean ± SD. Different letters represent significant differences between L-theanine vs. control at One-Way ANOVA *p < 0.05, **p < 0.001.

4. Discussion

In this study, we examined the effect of L-theanine on animal behaviour to detect its impact on cognitive performance. A maze test is used to elevate the level of anxiety and stress in mice, as by measuring the average completion time, we can know the degree of anxiety, and hence we can know the effectiveness of the treatment. According to Ogawa et al. (2018), the longer time spent by mice inside the maze indicates lower anxiety levels.

The primary aim of the current study was to assess the independent and interactive effects of L-theanine on the central nervous system. Our results here demonstrate that, compared to control, L-theanine has an antianxiety effect in a dose-dependent manner. L-theanine was shown to be easily absorbed in the intestinal tract and to have physiological activities that were similar to those of other amino acids, as it can be absorbed and reach brain tissue within 30 min of oral administration (Raj Juneja et al., n.d.). Vuong et al. (2011) found that cognitive function was significantly enhanced by consuming 50 mg of L-theanine in 100 ml of water. In behavioural testing, L-theanine at a dose of 2 mg/kg/day for 21 days increased serotonin and dopamine, which reduced symptoms of depression (Shen et al., 2019). Both the cerebellum and the hippocampus have a role in anxiety-related actions (Wang et al., 2022), whereas the cerebellar granule cells (CGCs) have a role in the management of anxious behaviour (Jiang et al., 2023).

In different subfields of behavioral neuroscience, the Morris Water Maze test has become one of the most commonly utilized research methods. The water maze test showed that appropriate dosages of L-theanine decrease the mistake number and shorten the time for mice to escape the maze (Haskell et al., 2008). L-Theanine (180 mg/day) was administered to weanling male Wistar rats for four months to investigate its effect on memory and learning ability. Compared to the control group, the theanine group had a higher frequency of accurate responses. In addition to relatively good reaction frequency in a series of memory and learning ability tests, a group administered L-theanine solution (1 g/100 ml water) for a long period (five months) (Raj Juneja et al., n.d.).

The cerebellum's primary function in spatial learning is to control the procedural aspects of the task. Rather than knowing an object's spatial location, the cerebellum may be involved in procedures that are required to find it in space. Specific deficits were found in animals with damage to the hippocampus, striatum, basal forebrain, cerebellum, and several neocortical areas, and specific roles in MWM performance have been proposed for these regions (D’Hooge and De Deyn, 2001).

In this study, L-theanine was used at different concentrations in the microexplant cultures of P3–P4 mouse cerebella to evaluate its effect on granule cell migration. Because of their inbred genetic background and extensive usage in neurodevelopmental research, which guarantees consistency and repeatability in neural culture systems, C57BL/6 N mice were employed. At postnatal days 3–4 (P3–P4), when cerebellar granule cell migration and proliferation are at the greatest level, cerebellar tissues were collected. For in vitro investigations of CGC migration and signaling, this age range is generally acknowledged (Hatten, 1985; Komuro and Rakic, 1992).

This culture system mimics the in vivo migration behaviour where the majority of CGCs migrate within the first three postnatal days, the time required for the cells to complete the migration (Komuro and Yacubova, 2003a). The granule cells, originating from progenitor cells in the EGL, undergo significant differentiation and migration in the first two postnatal weeks in rodents (Xu et al., 2013). Changes in local environmental cues, such as cell adhesion molecules, influence granule cell shape and migration rate, with gene expression playing a key role in regulating these processes. The CGCs move from the top of the EGL through the ML and Purkinje cell layer (PCL) to their final position in the IGL within approximately two days (average 51 h). The granule cells take an average of 25.0 h to reach their final location in the EGL, 9.8 h in the ML, 5.2 h in the PCL, and 11.1 h in the IGL (Komuro and Yacubova, 2003b), thereby explaining the experimental timeline within the first 48 h of explant isolation. Numerous studies have linked L-theanine's interactions with glutamate receptors to its neuroprotective properties. Furthermore, L-theanine interacts with a variety of glutamate receptors because it shares structural similarities with the neurotransmitter glutamate, including NMDA, AMPA, and kainate receptors. L-theanine dramatically improves cognitive performance, even at low doses. It is rapidly absorbed and penetrates the brain tissue, thereby affecting the glutamate receptors associated with anxiety, learning, and memory, that showed an enhancing effect on cognitive performance (Dodd et al., 2015). L-theanine as shown to affect motor sensor ability, which influences the result of the maze test, increasing the grooming time, which indicates exploratory behaviour, decision-making, or cognitive conflict. L-theanine can promote a state of relaxation without causing drowsiness. The effect of L-theanine on CGC differentiation and migration is influenced by various extracellular factors. This differentiation affects neurotransmitter uptake by selectively inhibiting the reuptake of glutamine and glutamate (Adhikary and Mandal, 2017). Some studies have demonstrated that the CGCs, which are crucial for neuronal development, exhibit increased migration with low micromolar concentrations of L-theanine (Komuro and Yacubova, 2003a; Komuro et al., 2021).

In this study, we studied a range of nano- and micromolar concentrations of L-theanine in comparison to other studies that only tested materials up to 10 µM concentration (Y. Komuro et al., 2021; Kunimoto and Suzuki, 1997; Lakatos et al., 2020). Consistent with the galanin study, which demonstrated that CGCs are highly affected by 1 µM concentration (Komuro et al., 2021), L-theanine, also at 1 µM concentration in the current study, demonstrated the highest impact on the CGC migration distance. While previous studies have investigated the impact of L-theanine on CGC migration at high concentrations, this study investigates its effect at low concentrations. Our findings show that L-theanine concentrations have a dose-dependent effect on CGC migration, with the greatest effect being shown at 1 μM L-theanine.

These results demonstrate the potential of L-theanine as an inducer of CGC migration at a concentration of 1 µM, highlighting its function in modifying CGC migration. Future research should concentrate on elucidating the molecular processes by which L-theanine generates these effects and assessing their long-term consequences on behaviour and cerebellar function. These findings suggest that L-theanine may be used in clinical settings to improve neurodevelopment and address related disorders.

Although L-theanine's neurotransmitter-modulating properties suggest a potential role in migration, there is insufficient evidence to support its direct effects on the proteins or receptors involved in migration. In the context of migration, its effects on intracellular signalling, oxidative stress, and the glutamate and γ-aminobutyric acid (GABA) pathways need more investigation. Through the regulation of glutamate receptors, which impacts signalling, L-theanine may cause CGC migration (Bajaj et al., 2021). Further research is required to clarify the mechanisms, particularly focusing on downstream signalling pathways and receptors that are specific to migration. Further behavioural experiments will hopefully confirm the beneficial actions of L-theanine in vivo and present a therapeutic strategy to alleviate the symptoms of various neurological/psychiatric diseases.

5. Conclusion

As a conclusion of our research, L-theanine improved anxiety symptoms in mice after a single dose of L-theanine at the concentration of 5 mg/ml. L-theanine significantly induced CGC migration at a concentration of 1 µM. These findings contribute to the multifaceted health benefits and therapeutic potential of L-theanine, highlighting its importance in current research aimed at exploring its comprehensive pharmacological effects. Further research is required to fully understand the mechanism through which L-theanine promotes CGC migration, which reflects to proper cognitive performance.

6. Limitations and future directions

The results of the maze test may be affected by individual differences in motivation or stress levels, which were not fully taken into consideration in this investigation. Test results could have varied depending on uncontrolled conditions like background noise or lighting. Because the maze test focuses on elevating spatial memory and learning, it may overlook other cognitive domains that are relevant to the research. The responses of other mice strains or female mice may not be well reflected using ddY male mice in this work due to potential genetic and hormonal differences. This study used only male mice to minimize hormonal variability, which may limit the generalizability of the findings across sexes. Future studies will investigate sex-dependent differences in response to L-theanine. Male ddY mice were used due to their suitability for behavioral tests like the Y-maze and Morris water maze. This outbred strain, common in Japan, exhibits good health, sensitivity to drugs, and moderate baseline cognition making it ideal for assessing cognitive changes. Prior studies have successfully used ddY mice to evaluate the effects of L-theanine on learning and memory (Wang et al., 2022).

The effects of L-theanine were assessed following a single administration, which ignores any potential cumulative or long-term effects of L-theanine. This study focused on single-dose L-theanine effects, capturing only acute responses. However, long-term administration may induce broader changes in behavior and neuroplasticity (Unno et al., 2013, Nobre et al., 2008). warranting further research on its impact on cerebellar development and cell migration. However, theanine alone in a 0.05 mg/ml concentration has a significant effect on elevating the ability of learning and memory. Although there are factors such as the work in which the MWM has been used, variances in apparatus or training technique, and research instruments that all influence MWM performance, swimming speed can be affected by factors such as body weight, physical development, and age. MWM performance can be affected by gender, as well as the strain/species of the animal subjects. Males consistently outperform females, and this cannot be attributed solely to differences in muscle strength or endurance. Finally, multiple studies have shown that stressed, unwell, undernourished, or elderly animals have serious impairments in spatial learning (D’Hooge and De Deyn, 2001).

Future studies should combine behavioural assays with molecular analysis, such as neurotransmitter profiling or brain imaging, to elucidate the process behind L-theanine action. Incorporating tests like the elevated plus maze, open field test, and social interaction paradigms may allow for a more comprehensive investigation of L-theanine’s effects. Future studies should examine intracellular signaling cascades, including calcium signaling or Mitogen activated protein kinases (MAPK) pathways, to have a better understanding of how L-theanine influences CGC migration. Live-cell imaging combined with fluorescently tagged proteins may provide real-time insights into cytoskeletal remodeling during CGC migration.

Author contributions

Conceptualization: [Mai Ibrahim, Nishio Masahiro], Methodology: [Ozeki Makoto, Abe Aya], Formal analysis and investigation: [Mai Ibrahim], Writing - original draft preparation: [Mai Ibrahim, Kuriya Kenji], Writing - review and editing: [Hayato Umekawa], Resources: [Matsuda Tomoko], Supervision: [Nishio Masahiro]

CRediT authorship contribution statement

Ozeki Makoto: Resources. Kuriya Kenji: Methodology. Matsuda Tomoko: Methodology, Formal analysis. Mai Ibrahim: Writing – original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Masahiro Nishio: Writing – review & editing, Supervision, Project administration, Data curation, Conceptualization. Hayato Umekawa: Methodology, Formal analysis, Data curation. Abe Aya: Resources, Methodology.

Competing with ethical standards

All experimental procedures adhered to the Mie University guidelines for laboratory animals, (reference number 2022–5), approved by the Institutional Animal Care and Use Committee of Mie University, and complied with the governmental guidelines (the Japanese Association of Laboratory Animal Facilities of National University Corporations).

Funding

This work was supported by Japan Science and Technology Agency (JST); Support for Pioneering Research Initiated by the Next Generation (SPRING), Japan (grant number JPMJSP2137).

Declaration of Competing Interest

The authors declare that they have no conflict of interest

Acknowledgments

I would like to acknowledge the Graduate School of Bioresources, Mie University, and Japan Science and Technology Agency (JST); Support for Pioneering Research Initiated by the Next Generation (SPRING), Japan for providing financial support. I want to thank Taiyo Kagaku Co. Ltd., Japan for their experimental support with L-theanine powder and maze apparatus.

Data availability

All data generated or analyzed during this study are included in this published article.

References

  1. Adhikary R., Mandal V. Vol. 7. Hainan Medical University; 2017. L-theanine: A potential multifaceted natural bioactive amide as health supplement; pp. 842–848. (In Asian Pacific Journal of Tropical Biomedicine). [DOI] [Google Scholar]
  2. Bajaj S., Bagley J.A., Sommer C., Vertesy A., Nagumo Wong S., Krenn V., Lévi-Strauss J., Knoblich J.A. Neurotransmitter signaling regulates distinct phases of multimodal human interneuron migration. EMBO J. 2021;40(23):1–21. doi: 10.15252/embj.2021108714. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Borghesani Paul R., Peyrin Jean Michel, Klein Robyn, Rubin Joshua, Carter Alexander R., Schwartz Phillip M., Luster Andrew, Corfas Gabriel, Segal Rosalind A. BDNF stimulates migration of cerebellar granule cells. Development. 2002;129(6):1435–1442. doi: 10.1242/dev.129.6.1435. [DOI] [PubMed] [Google Scholar]
  4. Bryan J. Psychological effects of dietary components of tea: caffeine and L-theanine. Nutr. Rev. 2008;66(2):82–90. doi: 10.1111/j.1753-4887.2007.00011.x. [DOI] [PubMed] [Google Scholar]
  5. Butts T., Green M.J., Wingate R.J.T. Development of the cerebellum: simple steps to make a ‘little brain. Development (Cambridge) 2014;141(21):4031–4041. doi: 10.1242/dev.106559. [DOI] [PubMed] [Google Scholar]
  6. D’Hooge R., De Deyn P.P. Applications of the Morris water maze in the study of learning and memory. Brain Res. Rev. 2001;36(1) doi: 10.1016/S0165-0173(01)00067-4. [DOI] [PubMed] [Google Scholar]
  7. Dodd F.L., Kennedy D.O., Riby L.M., Haskell-Ramsay C.F. A double-blind, placebo-controlled study evaluating the effects of caffeine and L-theanine both alone and in combination on cerebral blood flow, cognition and mood. Psychopharmacology. 2015;232(14):2563–2576. doi: 10.1007/s00213-015-3895-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dramard V., Kern L., Hofmans J., Rème C.A., Nicolas C.S., Chala V., Navarro C. Effect of l-theanine tablets in reducing stress-related emotional signs in cats: an open-label field study. Ir. Vet. J. 2018;71(1) doi: 10.1186/s13620-018-0130-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gilbert, C.Gilbert, C. 2014. THE EFFECT OF TEA AND ITS CONSTITUENT L-THEANINE ON ANXIETY: A REVIEW OF THE LITERATURE.
  10. Haskell C.F., Kennedy D.O., Milne A.L., Wesnes K.A., Scholey A.B. The effects of l-theanine, caffeine and their combination on cognition and mood. Biol. Psychol. 2008;77(2):113–122. doi: 10.1016/j.biopsycho.2007.09.008. [DOI] [PubMed] [Google Scholar]
  11. Hatten M.E. Neuronal regulation of astroglial morphology and proliferation in vitro. J. Cell Biol. 1985;100(2):384–396. doi: 10.1083/jcb.100.2.384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hidese S., Ogawa S., Ota M., Ishida I., Yasukawa Z., Ozeki M., Kunugi H. Effects of L-Theanine administration on stress- related symptoms and cognitive functions in healthy adults: a randomized controlled trial. Nutrients. 2019;11(10) doi: 10.3390/nu11102362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jiang Y., Cameron D.B., Hack N.J., Sluiter A.A., Balfizs R., Therapies A., Dasdelen M.F., Er S., Kaplan B., Celik S., Beker M.C., Orhan C., Tuzcu M., Sahin N., Sahin K., Kilic E., Ratih K., Lee Y., Chung K., Usha R. Excess cerebellar granule neurons induced by the absence of p75NTR during development elicit social behavior deficits in mice. Neurosci. Lett. 2023;16(2):1–13. doi: 10.1016/S0304-3940(97)00273-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kakuda T., Nozawa A., Sugimoto A., Niino H. Inhibition by Theanine of Binding of [3H]AMPA, [3H]Kainate, and [3H]MDL 105,519 to Glutamate Receptors. Biosci. Biotechnol. Biochem. 2002;66(12):2683–2686. doi: 10.1271/bbb.66.2683. [DOI] [PubMed] [Google Scholar]
  15. Kokubo M., Nishio M., Ribar T.J., Anderson K.A., West A.E., Means A.R. BDNF-mediated cerebellar granule cell development is impaired in mice null for CaMKK2 or CaMKIV. J. Neurosci. 2009;29(28):8901–8913. doi: 10.1523/JNEUROSCI.0040-09.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Komuro Y., Galas L., Morozov Y.M., Fahrion J.K., Raoult E., Lebon A., Tilot A.K., Kikuchi S., Ohno N., Vaudry D., Rakic P., Komuro H. The role of galanin in cerebellar granule cell migration in the early postnatal mouse during normal development and after injury. J. Neurosci. 2021;41(42):8725–8741. doi: 10.1523/JNEUROSCI.0900-15.2021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Komuro H., Rakic P. Selective role of N-type calcium channels in neuronal migration. Science. 1992;257(5071):806–809. doi: 10.1126/science.1323145. [DOI] [PubMed] [Google Scholar]
  18. Komuro H., Yacubova E. Recent advances in cerebellar granule cell migration. Cell. Mol. Life Sci. 2003;60:1084–1098. doi: 10.1007/s00018-003-2248-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Komuro H., Yacubova E. Recent advances in cerebellar granule cell migration. Cell. Mol. Life Sci. 2003;60(6):1084–1098. doi: 10.1007/s00018-003-2248-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Krämer D., Minichiello L. Cell culture of primary cerebellar granule cells. Methods Mol. Biol. 2010;633:233–239. doi: 10.1007/978-1-59745-019-5_17. [DOI] [PubMed] [Google Scholar]
  21. Kuhad A., Chopra K. Curcumin attenuates diabetic encephalopathy in rats: behavioral and biochemical evidences. Eur. J. Pharmacol. 2007;576(1–3):34–42. doi: 10.1016/j.ejphar.2007.08.001. [DOI] [PubMed] [Google Scholar]
  22. Kuhad A., Chopra K. Effect of sesamol on diabetes-associated cognitive decline in rats. Exp. Brain Res. 2008;185(3):411–420. doi: 10.1007/s00221-007-1166-y. [DOI] [PubMed] [Google Scholar]
  23. Kunimoto M., Suzuki T. Migration of granule neurons in cerebellar organotypic cultures is impaired by methylmercury. Neurosci. Lett. 1997;226(3):183–186. doi: 10.1016/S0304-3940(97)00273-5. [DOI] [PubMed] [Google Scholar]
  24. Lakatos P.P., Vincze I., Nyariki N., Bagaméry F., Tábi T., Szökő É. The effect of L-theanine and S-ketamine on D-serine cellular uptake. Biochim. Et. Biophys. Acta Proteins Proteom. 2020;1868(10) doi: 10.1016/j.bbapap.2020.140473. [DOI] [PubMed] [Google Scholar]
  25. Li M.Y., Liu H.Y., Wu D.T., Kenaan A., Geng F., Li H.Bin, Gunaratne A., Li H., Gan R.Y. Vol. 9. Frontiers Media S.A.; 2022. L-Theanine: A unique functional amino acid in tea (Camellia sinensis L.) with multiple health benefits and food applications. (In Frontiers in Nutrition). [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Morris R.G.M., Garrud P., Rawlins J.N.P., O’Keefe J. Place navigation impaired in rats with hippocampal lesions. Nature. 1982;297(5868):681–683. doi: 10.1038/297681a0. [DOI] [PubMed] [Google Scholar]
  27. Nathan P.J., Lu K., Gray M., Oliver C. The neuropharmacology of L-theanine(N-ethyl-L-glutamine): a possible neuroprotective and cognitive enhancing agent. J. Herb. Pharmacother. 2006;6(2):21–30. doi: 10.1300/J157v06n02_02. [DOI] [PubMed] [Google Scholar]
  28. Nobre A.C., Rao Phd A., Owen G.N. L-theanine, a natural constituent in tea, and its effect on mental state. Asia Pac. J. Clin. Nutr. 2008;17(S1) [PubMed] [Google Scholar]
  29. Ogawa S., Ota M., Ogura J., Kato K., Kunugi H. Effects of l-theanine on anxiety-like behavior, cerebrospinal fluid amino acid profile, and hippocampal activity in Wistar Kyoto rats. Psychopharmacology. 2018;235(1):37–45. doi: 10.1007/s00213-017-4743-1. [DOI] [PubMed] [Google Scholar]
  30. Paredes M.F., Sorrells S.F., Garcia-Verdugo J.M., Alvarez-Buylla A. Brain size and limits to adult neurogenesis. J. Comp. Neurol. 2016;524(3):646–664. doi: 10.1002/cne.23896. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Pawlak R., Magarinos A.M., Melchor J., McEwen B., Strickland S. Tissue plasminogen activator in the amygdala is critical for stress-induced anxiety-like behavior. Nat. Neurosci. 2003;6(2):168–174. doi: 10.1038/nn998. [DOI] [PubMed] [Google Scholar]
  32. Raj Juneja, L., Chu, D.-C., Okubo, T., Nagato, Y., & Yokogoshi, H. n.d. L-theanine a unique amino acid of green tea and its relaxation effect in humans. 10.1016/S0924-2244(00)00031-5. [DOI]
  33. Robbins T.W., Murphy E.R. Behavioural pharmacology: 40+ Years of progress, with a focus on glutamate receptors and cognition. Trends Pharmacol. Sci. 2006;27(3 SPEC. ISS.):141–148. doi: 10.1016/j.tips.2006.01.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sebih F., Rousset M., Bellahouel S., Rolland M., De Jesus Ferreira M.C., Guiramand J., Cohen-Solal C., Barbanel G., Cens T., Abouazza M., Tassou A., Gratuze M., Meusnier C., Charnet P., Vignes M., Rolland V. Characterization of l -Theanine excitatory actions on hippocampal neurons: toward the generation of novel N-methyl- d -aspartate receptor modulators based on its backbone. ACS Chem. Neurosci. 2017;8(8):1724–1734. doi: 10.1021/acschemneuro.7b00036. [DOI] [PubMed] [Google Scholar]
  35. Shen M., Yang Y., Wu Y., Zhang B., Wu H., Wang L., Tang H., Chen J. L-theanine ameliorate depressive-like behavior in a chronic unpredictable mild stress rat model via modulating the monoamine levels in limbic–cortical–striatal–pallidal–thalamic-circuit related brain regions. Phytother. Res. 2019;33(2):412–421. doi: 10.1002/ptr.6237. [DOI] [PubMed] [Google Scholar]
  36. Takeda A., Sakamoto K., Tamano H., Fukura K., Inui N., Suh S.W., Won S.J., Yokogoshi H. Facilitated neurogenesis in the developing hippocampus after intake of theanine, an amino acid in tea leaves, and object recognition memory. Cell. Mol. Neurobiol. 2011;31(7):1079–1088. doi: 10.1007/s10571-011-9707-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Tuzcu M., Baydas G. Effect of melatonin and vitamin E on diabetes-induced learning and memory impairment in rats. Eur. J. Pharmacol. 2006;537(1–3):106–110. doi: 10.1016/j.ejphar.2006.03.024. [DOI] [PubMed] [Google Scholar]
  38. Unno K., Iguchi K., Tanida N., Fujitani K., Takamori N., Yamamoto H., Ishii N., Nagano H., Nagashima T., Hara A., Shimoi K., Hoshino M. Ingestion of theanine, an amino acid in tea, suppresses psychosocial stress in mice. Exp. Physiol. 2013;98(1):290–303. doi: 10.1113/expphysiol.2012.065532. [DOI] [PubMed] [Google Scholar]
  39. Vuong Q.V., Bowyer M.C., Roach P.D. L-Theanine: properties, synthesis and isolation from tea. J. Sci. Food Agric. 2011;91(11):1931–1939. doi: 10.1002/jsfa.4373. [DOI] [PubMed] [Google Scholar]
  40. Wang L., Brennan M., Li S., Zhao H., Lange K.W., Brennan C. Vol. 11. Elsevier B.V; 2022. How does the tea L-theanine buffer stress and anxiety; pp. 467–475. (In Food Science and Human Wellness). [DOI] [Google Scholar]
  41. Williams J.L., Everett J.M., D’Cunha N.M., Sergi D., Georgousopoulou E.N., Keegan R.J., McKune A.J., Mellor D.D., Anstice N., Naumovski N. The effects of green tea amino acid L-theanine consumption on the ability to manage stress and anxiety levels: a systematic review. Plant Foods Hum. Nutr. 2020;75(1):12–23. doi: 10.1007/s11130-019-00771-5. [DOI] [PubMed] [Google Scholar]
  42. Xu H., Yang Y., Tang X., Zhao M., Liang F., Xu P., Hou B., Xing Y., Bao X., Fan X. Bergmann glia function in granule cell migration during cerebellum development. Mol. Neurobiol. 2013;47(2):833–844. doi: 10.1007/s12035-013-8405-y. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

All data generated or analyzed during this study are included in this published article.

Articles from IBRO Neuroscience Reports are provided here courtesy of Elsevier

RESOURCES