Effects of single-dose L-theanine on motor cortex excitability

L-theanine (N⁵-ethyl-L-glutamine) is one of the primary psychoactive components uniquely contained in green tea (Camellia sinensis). It structurally resembles L-glutamine and crosses the blood-brain barrier in a dose-dependent manner (Sakamoto et al., 2019). Wakabayashi et al. demonstrated dose-dependent antipsychotic-like and possibly antidepressant-like effects of L-theanine in rodents. They posited it to be a partial N-methyl-D-aspartate receptor (NMDA-R) agonist, due to its ability of increasing Ca²⁺ influx in rodent neurons- an effect which was blocked by the competitive NMDA-R antagonist MK-801 (Wakabayashi et al., 2012). Clinical trials have suggested that L-theanine at 200–400 mg daily dosing for 4–8 weeks as an adjunctive therapy improves anxiety, depressive and psychotic symptoms with minimal side effects (Sakamoto et al., 2019). Glutamatergic and γ-aminobutyric acid (GABA)-ergic systems assessed by motor cortex excitability using transcranial magnetic stimulation (TMS) are aberrant in these psychiatric disorders (Bunse et al., 2014). However, we do not know if the improvement of the symptoms from L-theanine is associated with correction of the aberrance. Even in healthy humans, the neurophysiological effects of L-theanine on the glutamatergic and GABAergic systems have not been determined.

Paired-pulse transcranial magnetic stimulation (ppTMS) protocols measuring intracortical facilitation (ICF), short- and long-interval intracortical inhibition (SICI, LICI) offer unique opportunities to probe the human motor cortex intracortical excitability in vivo (Ziemann et al., 2015). We proposed a study to use these measurements to investigate how L-theanine changes the glutamatergic (indicated by ICF) and GABAergic systems (indicated by SICI for GABA_A-R and LICI for GABA_B-R) in the primary motor cortex (M1) in healthy subjects. The study was approved by Butler Hospital Institutional Review Board, and registered on clinicaltrials.gov (NCT04749745). Given the potential NMDA-R agonistic effects of L-theanine, we hypothesized that a single-dose would enhance ICF acutely and may attenuate SICI and/or LICI.

This was a placebo-controlled, randomized double-blind crossover study in 10 healthy adult subjects (5 males, 5 females, age 25–36 years old). All participants provided informed consent prior to any research procedures, after screening by Structured Clinical Interview for DSM-V (SCID-V-NP [non-patient]) to confirm absence of neuropsychiatric disorders and contraindications for TMS. Subjects were randomized to 400 mg oral single-dose of L-theanine (Nootropics Depot, AZ) or matching placebo (cellulose capsule) on 2 separate visits at least 72-hours apart (Figure-1A). All 10 participants received both conditions in random order (6 received placebo; 4 received L-theanine in the first visit). The ppTMS protocols were performed prior to, and 30 min (Sakamoto et al., 2019) after drug administration using a Mag&More PowerMAG stimulator and PMD70-pCool figure-8 coil (Munich, Germany). We ensured all ppTMS pulses were delivered to the left motor cortex (M1) using Brainsight neuronavigation (Rogue Research, Quebec, Canada). Resting motor threshold (rMT) was determined as the lowest stimulator intensity to elicit 50 μV motor-evoked potential (MEP) in ≥5/10 trials on surface electromyography over the right abductor pollicis brevis (APB).

Fig. 1.

The Effects of L-Theanine on Motor Cortex Intracortical Excitability Measured by SICI, ICF and LICI. (A) Study Design. (B–D) Comparison between pre-to-post-drug SICI change (ΔSICI), ICF change (ΔICF) and LICI change (ΔLICI), respectively under L-theanine Condition and Placebo Condition. Mean ± SE ΔSICI_L-theanine = 0.059 ± 0.028 vs. ΔSICI_Placebo = 0.005 ± 0.060, p = 0.450, Cohen’s d = 0.35. The black lines connect the same subjects between two conditions. The orange line represents mean ΔSICI under L-theanine condition and placebo condition. Mean ± SE ΔICF_L-theanine = 0.073 ± 0.073 vs. ΔICF_Placebo = −0.341 ± 0.176, *p = 0.016, Cohen’s d = 1.02. The black lines connect the same subjects between two conditions. The blue line represents mean ΔICF under L-theanine condition and placebo condition. Mean ± SE ΔLICI_L-theanine = 0.145 ± 0.100 vs. ΔLICI_Placebo = −0.068 ± 0.053, *p = 0.037, Cohen’s d = 0.91. The black lines connect the same subjects between two conditions. The red line represents mean ΔLICI under L-theanine condition and placebo condition. Δ value > 0 means an increase in SICI, ICF or LICI value; Δ value < 0 means a decrease in SICI, ICF or LICI value. Note that an increase in ICF value indicates enhanced NMDA-R mediated intracortical facilitation; whereas an increase in SICI or LICI value indicates attenuation of GABA_A-R or GABA_B-R mediated intracortical inhibition, respectively. Abbreviations: ppTMS = Paired-pulse Transcranial Magnetic Stimulation, SICI = Short-interval Intracortical Inhibition, ICF = Intracortical Facilitation, LICI = Long-interval Intracortical Inhibition, SE = Standard Error of the Mean, NMDA-R = N-methyl-D-aspartate receptor, GABA_B-R = γ-aminobutyric acid-_B receptor.

SICI and ICF protocols were conducted with conditioning stimulus (CS) at 80%-rMT and test stimulus (TS) at 120%-rMT. The inter-stimulus intervals (ISIs) for SICI were 2 ms and 5 ms; for ICF were 10 ms and 20 ms. LICI was measured with CS at 120%-rMT and TS at 120%-rMT with ISIs of 100 ms and 200 ms (Ziemann et al., 2015). 24 trials of each ISI-condition were collected (Chang et al., 2016). We set the intertrial interval (ITI) at 3–5 sec. The MEPs of SICI, ICF and LICI were averaged between 2 ms and 5 ms (48 trials), 10 ms and 20 ms (48 trials), 100 ms and 200 ms (48 trials), respectively, as we did not observe differential effects of L-theanine between each pair of ISIs. Given that we conducted all trials (including SICI, ICF, LICI and unconditioned pulse) in one randomized block, unconditioned MEP was the average of 24 trials of single pulse at 120%-rMT, and 48 trials from the CS in LICI protocol. SICI, ICF and LICI values were calculated by the ratio of conditioned MEP / unconditioned MEP. We used Wilcoxon signed-rank tests to compare the baseline-to-post-drug changes (Δ = post-drug value minus pre-drug value) of SICI, ICF and LICI between placebo and L-theanine conditions.

Compared to matching placebo, 400 mg single dose L-theanine elicited significantly higher post-pre drug change (Δ) of ICF (Mean ± SE ΔICF_L-theanine = 0.073 ± 0.073 vs. ΔICF_Placebo = −0.341 ± 0.176, p = 0.016, Cohen’s d = 1.02, Fig. 1C) and LICI (Mean ± SE ΔLICI_L-theanine = 0.145 ± 0.100 vs. ΔLICI_Placebo = −0.068 ± 0.053, p = 0.037, Cohen’s d = 0.91, Fig. 1D) within each individual. No significant difference was found for SICI (Fig. 1B). L-theanine did not change unconditioned MEPs (or baseline rMT) differently than placebo (Table 1). No adverse effects from L-theanine were observed. The subjects were unable to guess the condition of drug (placebo vs L-theanine) after each session of experiment more accurately than random.

Table 1.

Results on Unconditioned MEPs, Side Effects and Subject Perception of Drug Condition.

	L-Theanine	Placebo	P-value*
Pre-Drug Unconditioned MEPs (mV, Mean ± SE)	0.877 ± 0.174	0.798 ± 0.229	0.375
Post-Drug Unconditioned MEPs (mV, Mean ± SE)	0.960 ± 0.247	0.852 ± 0.172	0.557
Pre-to-post-Drug Change (Δ) of Unconditioned MEPs (mV, Mean ± SE)	0.082 ± 0.163	0.054 ± 0.093	0.557
Side Effects	None observed	None observed	N/A
Number of Subjects who Guessed Condition Correctly	4/10	4/10	N/A

MEPs = Motor Evoked Potentials, mV = microvolts, SE = Standard Error of the Mean, N/A = Not Applicable.

^*Comparisons were made using Wilcoxon Signed-rank Test.

Although limited by the small sample size, this study is the first to advance our knowledge regarding the effects of an NMDA-R agonist on motor cortex excitability, while several studies on the effects of NMDA-R antagonists, including dextromethorphan, memantine, and riluzole, have been reported (Ziemann et al., 2015). This is also the first report showing the effects of L-theanine, a substance of increasing therapeutic interest for anxiety, affective and psychotic disorder, on brain excitability.

The results suggest that a single dose of L-theanine may enhance NMDA-R mediated intracortical facilitation and attenuate GABA_B-R mediated intracortical inhibition in the human primary motor cortex. The observation that L-theanine did not change SICI while impacting LICI may represent the inhibitory effect of LICI on SICI, mediated by presynaptic GABA_B-R (Chen, 2004). With the caveat that the results need to be replicated, we hope these data could lay the groundwork for future investigations into the therapeutic mechanisms of L-theanine in neuropsychiatric disorders.