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Green tea contains caffeine and catechins (flavonoids and antioxidants), but it also contains L-Theanine. L-Theanine is one of green tea’s active components. In this article, we will learn more about this often overlooked active ingredient. Specifically, the ways that L-Theanine can impact anxiety, behavior, and cognition.
What is L-Theanine?
Gamma-ethylamino-L-glutamic acid (L-Theanine) is almost exclusively found in Camellia sinensis, an evergreen tea plant. L-Theanine makes up about 50% of green tea’s free amino acids. As an amino acid, L-theanine is non-proteinous and water-soluble.
L-theanine is a biologically active compound that is able to pass through the blood-brain barrier, thus easily impacting the brain neurochemically.
In the brain, L-Theanine will act as a neurotransmitter, since it is a derivative of L-glutamic acid which is the brain’s major excitatory neurotransmitter. L-Theanine affects many of the brain’s neurotransmitters. It can increase dopamine concentrations in the hippocampus and striatum while decreasing norepinephrine levels in the brain. Also, some studies report that serotonin and GABA levels are increased due to L-Theanine. L-Theanine has also been reported to be able to reduce glutamate reuptake by means of inhibiting the glutamate transporter.
L-Theanine was first identified in 1949 and by 1950 it was successfully isolated from green tea. Since then, literature and experiments focusing both on humans and animals have exponentially grown showing L-Theanine’s many therapeutic and pharmacological uses, such as to reduce anxiety due to its naturally anxiolytic properties.
In this article, we will go over a few research findings and experiments, as well as the methods (animal models and mazes) used, which established L-Theanine’s neuroprotective and enhancing properties on cognition and behavior.
L-Theanine Reduces Anxiety in an Animal Model of Anxiety
Although research is establishing a relationship between L-Theanine intake and decreased anxiety levels, there is still insufficient evidence for this supplement to become a medicine that is prescribed by doctors as a part of their practice. Therefore, L-Theanine is taken as a dietary supplement by people that are self-medicating, in hopes of reducing their anxiety levels. Before L-Theanine can be prescribed officially by doctors, many more studies using humans and double-blind clinical trials need to occur. Currently, animal research is becoming incredibly useful in demonstrating the mechanisms and effects of phenibut on anxiety-related behaviors.
Research shows that L-Theanine supplementation was able to decrease heart rate and salivary immunoglobulin A responses in participants which underwent an acute stress task.
Furthermore, tea drinkers report that tea makes them feel relaxed without a sense of drowsiness. Another study confirmed this by giving young participants 50 mg of L-Theanine and measuring their brain waves with an electroencephalogram (EEG). Compared to the control group which received water, the treatment group had a greater level of alpha activity when the participants were in a resting state (eyes closed). Since alpha waves are associated with attention and alertness, these findings support the claim that tea (thus, L-Theanine) create a relaxed but attentive mental state.
L-Theanine has been also been shown to have an anxiolytic effect in behavioral studies using animal models of anxiety.
A group of scientists demonstrated this by conducting behavioral experiments in Wistar Kyoto rats which are known to be an animal model of anxiety and depressive disorders.
L-Theanine (0.4 mg kg-1 day-1 or saline administration (for the control)) was given for 10 days. On day 8 of the supplementation schedule, behavior was measured based on performance in the Elevated Plus-Maze, a maze that is commonly used to assess levels of anxiety in rodents.
The researchers found that the anxious rats given L-Theanine significantly spent more time in the maze’s open arms (on average 60 seconds) than the saline control group did (on average 20 seconds). Since spending time in the open arms is naturally anxiety provoking, the fact that the L-Theanine group had a longer time duration in the maze’s open arms means that L-Theanine has an anxiolytic effect on behavior.
L-Theanine Improves Cognition in Chronic Restraint Stress Model
The chronic restraint stress model has been shown to affect animals’ behavior, cognition, and physiology. The restraint stress model is established as being a convenient and easy method for inducing physical and psychological stress, useful for modeling the affective disorders precipitated by prolonged stress.
Prolonged stress is modeled by placing animals in maximum confinement (restraining the rodents in polypropylene tubes for 21 straight days for 8 consecutive hours per day). Due to this prolonged restraining, major stress is induced, thereby altering physiology and cognition. This is known as the chronic restraint stress-induced model.
In an experiment focusing on the effects of L-Theanine administration on stress, a group of researchers made use of the chronic restrained stress-induced model (CRS). In order to create multiple experimental conditions, some mice were given L-Theanine supplements 30 minutes prior to being placed in the tube for immobilization-induced stress and others were not.
To assess the impact of L-Theanine supplementation on learning and memory, the researchers used the Morris Water Maze for behavioral testing. The CRS mice were able to learn the maze just as well as the L-Theanine supplemented mice during the first few days of training, but began to show significant differences during the retention sessions. During the retention session, the mice were expected to have low transfer latency times, meaning that they could remember the location of the hidden platform well. Instead, the CRS mice had longer transfer latency times than the mice that had additional L-Theanine supplementation, indicating that the CRS mice were not able to remember spatial information as well as the CRS mice receiving L-Theanine. Also, the L-Theanine-treated mice spent significantly more amount of time in the target quadrant than the CRS mice, indicating stronger spatial memory skills.
When the rodents’ cortisol increment was analyzed, the CRS group had the highest levels while the L-Theanine-treated CRS group had significantly lower cortisol levels, indicating group differences in terms of the stress hormone. In terms of oxidative stress, lipid peroxidation was measured by assessing the malondialdehyde levels in the hippocampus and cerebral cortex. The L-Theanine-treated CRS mice had significantly lower levels than the CRS group, indicating that they did not have as much oxidative stress. Also, relative to the CRS group, the L-theanine-treated CRS group had a richer antioxidant profile, as shown by the higher levels of glutathione, superoxide dismutase, and catalase in both the hippocampus and cerebral cortex.
The combined findings, behavioral and physiological, show that L-Theanine has a major protective effect on cognition, memory, and the brain’s composition.
L-Theanine’s Antidepressant-like Effect in Mice
In addition to its protective role against chronic stress, L-Theanine has been demonstrated to have an antidepressant-like effect. Since typical pharmaceutical options are reported to have undesirable side effects and be effective only for a certain portion of patients, there is much need for more powerful, well-tolerated, and safe antidepressants. Therefore, L-Theanine has been gaining scientific attention as a potential candidate.
A group of researchers divided male kunming mice into several groups: control, clomipramine (a tricyclic antidepressant serving as a positive control) at 20 mg/kg, and L-Theanine at 1, 4, or 20 mg/kg for 10 consecutive days. Behavioral testing began 60 minutes after the last supplementation on day 10.
The researchers found that clomipramine and all of the L-Theanine conditions had significantly lower levels than the control group in terms of duration of immobility in the Forced Swim Test. In the Forced Swim Test, a mouse is placed in the water and kept there for 6 minutes. Behavior is measured during the last 4 minutes of the test and immobility is counted in terms of seconds whenever the mouse is completely still, not attempting to escape, and only making movements to keep its head above the water. Therefore, decreasing immobility time in the Forced Swim Test is a desirable outcome since immobility is associated with depressive-like behavior. L-Theanine supplementation for the course of 10 consecutive days at dosage levels 1, 4, or 20 mg/kg significantly reduced immobility time at each dose by 24.7%, 37.9%, and 38.4%, respectively. Clomipramine was also significantly lower than the control group, decreasing immobility time in the Forced Swim Test by 46.4%.
These positive findings carried over into the Tail Suspension Test in which clomipramine and all L-Theanine conditions once more significantly decreased the immobility times when compared with the control group. L-Theanine, at dosage levels 1, 4, or 20 mg/kg significantly reduced the length of immobility by 22.6%, 36.0%, and 39.9%, respectively. Clomipramine decreased the immobility time by 52.5% which was also significantly lower than the control group’s performance in the Tail Suspension Test.
In order to ensure that the increase of mobility is not attributable to hyperactivity, all of the mice were also subjected to the Open Field where general patterns of locomotor activity were quantified across the groups. There were no significant differences between the groups in terms of time spent crossing or rearing in the field between the control, the clomipramine and L-Theanine conditions.
To the researchers, the findings in the Forced Swim Test and the Tail Suspension Test combined with the locomotor activity performance results during the Open Field assessment, suggest that the positive results associated with L-Theanine are not attributable to a psychomotor-stimulant effect, but rather an antidepressant-like effect.
These behavioral results show that L-Theanine can significantly decrease immobility, a behavior commonly interpreted in terms of depression, in a similar way that clomipramine can.
Prenatal Exposure to Green Tea Extract Impacts Future Behavior
Prenatal exposure to green tea extract (GTE), in which L-Theanine is one of the major active components (in addition to caffeine and catechins), has been demonstrated to positively impact the offspring’s cognitive ability in mice, as demonstrated by one group of researchers.
The scientists designed three groups: a control group, and two groups receiving GTE at either 20 or 50 g/L concentrations. GTE was prepared with green tea which was purchased from the local market. Then, the dosages were prepared with either 20 or 50 g of green tea for 1000mL water. Boiling water was poured over the green tea leaves which were allowed to steep for 15 minutes. The concentrations were prepared on a daily basis, creating fresh concentrations every day. The mother was given 5mL of the respective concentration daily via oral intubation, 2.5mL in the morning and once again in the evening. The supplementation procedure began on the first day of pregnancy and lasted until postnatal day 22 (PD 22).
Then, on PD 25, in order to assess fear and anxiety in the pups, an Elevated Plus-Maze was used. Mice are placed in the middle of the maze, facing one of the maze’s open arms, and were observed closely for 5 minutes while they were supposed to be freely exploring the maze. Compared to the control group, the mice on GTE spent significantly more time in the open arms and had a greater number of entries in the open arms. Furthermore, males receiving either concentration of GTE had significantly fewer entries into the closed arms than the controls, an effect that was seen in females receiving only the low-concentration dose.
On PD 30, to measure learning and memory abilities, the researchers utilized a T-Maze, a maze that is T-shaped and has one long arm and a left and right arm. Before beginning this test, the pups are starved for 24 hours, receiving only water. Then, the mouse is placed in the T-Maze for an allotted time of 2 minutes where the food is located in the left arm, for example. After this initial trial, the mice are returned to their cage and the trial is repeated again 3 hours later. Several behavioral parameters are measured using the T-Maze, such as entrances and time spent in the food arm, as well as the latency time that it takes to reach the food. Behavioral analyses showed that the GTE mice had significantly more entries into the food arm and also had shorter latency time to arrive to the food than the control group did.
The combined behavioral findings, as found by using the T-Maze and the Elevated-Plus Maze, showed that GTE is able to enhance learning memory and learning, as well as produce anxiolytic effects, in offspring that were exposed to it prenatally.
L-Theanine Alters the Brain’s Reward Pathway
L-Theanine is also able to affect the brain’s reward pathway and decrease nicotine intake.
In an experiment studying the relationship between L-Theanine and the nicotine’s reward effects, as well as its underlying mechanisms, a group of scientists divided mice into the following groups:
- The control group injected with 0.9% saline subcutaneously
- The nicotine mice, receiving 0.5 mg kg-1 d-1
- The Th-L mice, receiving the low dose of L-Theanine, at 250 mg kg-1 d-1
- The Th-H mice, receiving the high dose of L-Theanine, at 500 mg kg-1 d-1
- The Th-L(N) mice injected with the low dose of L-Theanine and nicotine (at 250 mg kg-1 d-1 and 0.5 mg kg-1 d-1, respectively)
- The Th-H(N) mice injected with the high dose of L-Theanine and nicotine (at 500 mg kg-1 d-1 and 0.5 mg kg-1 d-1, respectively)
- The DHβE(N) mice, receiving nicotine and DHβE which antagonizes the behavioral effects of nicotine (at 0.5 mg kg-1 d-1 and 2.0 mg kg-1 d-1, respectively)
The L-Theanine and DHβE were given to the mice 15 minutes prior to their nicotine injection, thus acting as a form of pre-treatment.
Using the Conditioned Place Preference, a chamber which contains two distinct compartments where one of the compartments contains a vehicle solution and the other contains the drug, the researchers were able to observe significant behavioral differences across the groups. The mice that were given only nicotine spent significantly more time in the drug-paired compartment than the control group did. However, this preference was significantly inhibited when in mice that were assigned to the pre-treatment condition (receiving either DHβE or L-Theanine at either dose).
Then, the researchers ran a second experiment in which the mice did not receive pre-treatment, in order to rule out the possibility that L-Theanine can be protective only if it is taken prior to nicotine. So, supplementation was given only after the nicotine preference was induced. Still, the results remained unchanged. L-Theanine (and DHβE, as well) was still able to reduce the nicotine-induced preference for the drug-related chamber in the Conditioned Place Preference test, as shown the decreased time spent in the drug-related chamber.
Nicotine, just as other addictive drugs, affects the mesostriatal dopamine reward circuit which is a circuit composed of the prefrontal cortex, the ventral tegmental area, and the nucleus accumbens, and contains high levels of nicotine acetylcholine receptors (nAchRs). In addition to the behavioral changes associated with L-Theanine intake, this green tea compound was able to affect the composition of the brain’s reward pathway. The researchers conducted biochemical analyses on the experimental rodents’ brain tissue and found that L-Theanine inhibited the expression of nAChRs subunits, as revealed by Western blotting. The mice that received nicotine had higher levels of dopamine after two weeks than those that were pretreated with L-Theanine. Also, L-Theanine inhibited the upregulation of tyrosine hydroxylase (TH) which is a crucial enzyme for dopamine synthesis.
These results demonstrate that L-Theanine is able to affect mice on a cellular level which, in turn, becomes observable due to behavioral differences.
L-Theanine Reduces Aβ1-42-induced Memory Impairments
Amyloid β (Aβ)-induced neurotoxicity is one of the main pathological mechanisms of Alzheimer’s disease. It is widely acknowledged that Aβ is linked to the oxidative damage which characterizes the Alzheimer’s disease brain. Therefore, the antioxidant mechanisms of L-Theanine in green tea may be beneficial.
One experiment set out to find what L-Theanine’s inhibitory effect would do to the memory impairment and neuronal cell death in an Alzheimer’s disease model in mice. Prior to any model induction, eight-week-old male Slc:ICR mice were given L-Theanine which was added to their drinking water at 2 or 4 mg/kg, depending on condition. L-Theanine supplementation was given for 3 weeks, as the pretreatment, and for 2 additional weeks during behavioral tests.
Aβ1-42-induced memory impairment was created in mice by injecting Aβ1-42 (2 μg/mouse) into the third ventricle. The two control groups of the study included mice that received vehicle injections and mice that had Aβ1-42-induced memory impairment but were untreated and did not have L-Theanine added to their drinking water.
The first test which the mice were subjected to was the Morris Water Maze. In the Morris Water Maze, mice were trained daily for 6 days, but on the day before testing Aβ1-42 was injected into the mice. The mice that had pretreatment with L-Theanine were not as affected by the Aβ1-42-induced deficits as those without treatment. While the control mice (which received saline injections) had an escape latency of 10.2 seconds and a covered distance of 193.9 cm, the untreated Aβ1-42 mice moved 507.17 cm in 24.9 seconds prior to finding the hidden platform. This established that the animal model was working and that memory was impaired by Aβ1-42 injections. The L-Theanine-treated group, by comparison, had decreased escape latencies in a dose-dependent fashion. The 2 mg/kg condition covered 285 cm in 14.2 seconds prior to finding the hidden platform and the 4 mg/kg group traveled 248.7 cm in 12.4 seconds.
A few days later, the mice had to do the Passive/Avoidance Task which is also commonly referred to as the “Step Through Test.” In the Passive/Avoidance Task, mice are placed in an apparatus which has a well-lit compartment and a dark compartment. Due to their natural aversion to brightly lit spaces, mice will gravitate towards the dark compartment. Researchers, in order to test memory, will condition the mice to avoid the dark compartment by delivering a foot shock each time that the mouse enters into the dark compartment. Then, the step-through latency (how much time it takes for the mouse to re-enter the darker room) is measured and a higher latency is associated with better performance and memory abilities. In this experiment, the control groups had a latency of 26.8 seconds whereas the untreated Aβ1-42 mice had a latency of about 12.9 seconds. L-Theanine-treated Aβ1-42 mice had an increase in latency time in a dose-dependent fashion. The 2 mg/kg condition had a latency of 15.9 seconds and the 4 mg/kg condition of 24.9 seconds.
These findings show that administering L-Theanine for 5 weeks can significantly improve the memory of mice that were modeled to mimic Alzheimer’s disease. Such findings have serious (and promising) implications and should receive more attention from the research and health communities.
L-Theanine is able to affect cognition, affective behavior, and well-being, as demonstrated by numerous studies using various types of rodents. In fact, L-Theanine is able to enhance learning and memory, have anxiolytic and antidepressant-like effects, and alter the brain’s reward pathway.
Future studies should continue to examine this potent green tea’s active compound in the context of multiple animal models, including those that model anxiety disorders.
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