Category: Behavioral Pharmacology in Laboratory Animals

Wakabayashi et al.

ElectronicSupplementaryMaterial

Psychopharmacology

Category: Behavioral pharmacology in laboratory animals

Types of paper: OriginalInvestigation

Behavioral and molecular evidence for psychotropic effects in l-theanine

Chisato Wakabayashi1, Tadahiro Numakawa1, 2, Midori Ninomiya1, Shuichi Chiba1 and Hiroshi Kunugi*1, 2

1Department of Mental Disorder Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, 187-8502, Japan

2Core Research for Evolutional Science and Technology Program (CREST), Japan Science and Technology Agency (JST), Tokyo, 102-0075, Japan

* Address correspondence to Hiroshi Kunugi, M.D. Ph.D., Department of Mental Disorder Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1, Ogawahigashi, Kodaira, Tokyo, 187-8502, Japan, Tel & FAX: +81-42-346-1714, E-mail:

Including 7supplementary figures

Supplementary Figure S1. Effects of single administration of l-theanine in the open-field test (OFT). l-theanine (1, 10 mg/kg) or vehicle was injected 30 min before the test. (a) % center stay. (b) Total distance. The results are presented as means ± SEM (n = 5-9/group).

Supplementary Figure S2. Effects of single administration of l-theanine in the elevated plus-maze test (EPMT). l-theanine (4, 20, 100 mg/kg) or vehicle was injected 30 min before the test. (a) % open arm stay. (b) % open arm entry. (c) Total distance. (d) Total entry. The results are presented as means ± SEM (n = 7-8/group).

Supplementary Figure S3. Effects of single administration of l-theanine in the forced swim test (FST). Vehicle or l-theanine (4, 20, 100 mg/kg) was injected 30 min before the test. (a) Swimming time. (b) Immobilization time. (c) Climbing time. The results are presented as means ± SEM (n = 7-8/group).

Supplementary Figure S4.Subchronic administration of l-theanine does not affect the locomotor activityin the open-field test (OFT). Vehicle or l-theanine (0.4, 2, 10 mg/kg) was subchronically administered (i.p.) and then locomotor activity was analyzed in the OFT. (a) % center stay. (b) Total distance. The results are presented as means ± SEM (n = 9-12/group).

Supplementary Figure S5. Effects of subchronic administration of l-theaninein the elevated plus-maze test (EPMT). Vehicle or l-theanine (0.4, 2, 10 mg/kg) was subchronically administered (i.p.). (a) % open arm stay. (b) % open arm entry. (c) Total distance. (d) Total entry. The results are presented as means ± SEM (n = 9-12/group).

Supplementary Figure S6.Effects of subchronic administration of l-theanine on forced swimming test (FST). l-theanine (0.4, 2, 10 mg/kg, i.p.) or vehicle was subchronically administered every other day for 3 weeks. (a) swimming time, (b) immobilization time and (c) climbing time were analyzed. The results are presented as means ± SEM (n = 9-12/group). F = 6.5, df = 3, 37, p = 0.0012 for swimming (a).F = 6.7, df = 3, 37, p = 0.0010 for immobilization(b).Climbing activity tended to be increased by l-theanine in high dose administration (F = 2.33, df = 3, 37, p = 0.090) (c). *p < 0.05; **p < 0.01; ***p < 0.001 when compared with vehicle control (Dunnett’s test).

Supplementary Figure S7.Subchronic administration of l-theanine did not significantly change expression of BDNF, TrkB, p75, GluR1, and NR2B in the cortex. Vehicle or l-theanine (0.4, 2, 10 mg/kg) was subchronically administered (i.p.) and the hippocampus was removed. The equivalent amounts of total protein were applied to western blot analysis. Representative images of immunoblotting with anti-BDNF (a), anti-TrkB (b), anti-p75(c), anti-GluR1 (d), and anti-NR2A (e) antibodies were shown. The levels of BDNF, TrkB, p75, GluR1, and NR2A were quantified by densitometry, respectively. Data represent mean±SD (n=4, n indicates the number of mice for each experimental dose).Data were normalized to a level of vehicle control (without l-theanine).

1