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. Author manuscript; available in PMC: 2024 Jun 11.
Published in final edited form as: Neurosci Lett. 2023 Apr 25;807:137279. doi: 10.1016/j.neulet.2023.137279

L-theanine attenuates nicotine reward and withdrawal signs in mice

Yasmin Alkhlaif 1, Medhat El-Halawany 2, Wisam Toma 1, Abigail Park 1, Ayman K Hamouda 2, M Imad Damaj 1
PMCID: PMC10204724  NIHMSID: NIHMS1896528  PMID: 37105354

Abstract

Background:

L-theanine, 2-amino-4-(ethylcarbamoyl) butyric acid, an amino acid detected in green tea leaves, is used as a dietary supplement to attenuate stress and enhance mood and cognition. Furthermore, L-theanine induces anxiolytic effects in humans. Recently, L-theanine was reported to reduce morphine physical dependence in primates, suggesting the potential usefulness of L-theanine for drug dependence intervention.

Objective:

The aim of this study is to determine whether L-theanine attenuates nicotine-withdrawal (somatic and affective signs) and nicotine reward in mice. We also investigated the effects of L-theanine on nicotinic receptors binding and function.

Methods:

ICR male mice rendered dependent to nicotine through implanted subcutaneous osmotic minipumps for 14 days undertook precipitated nicotine withdrawal by mecamylamine on day 15. Anxiety-like behaviors using LDB, somatic signs observation and hot plate latency were assessed consecutively after treatment with L-theanine. Furthermore, we examined the effect of L-theanine on acute nicotine responses and nicotine conditioned reward in mice and on expressed nicotinic receptors in oocytes.

Key findings:

L-theanine reduced in a dose-dependent manner anxiety-like behavior, hyperalgesia and somatic signs during nicotine withdrawal. Also, L-theanine decreased the nicotine CPP, but it did not affect the acute responses of nicotine. Finally, L-theanine did not alter the binding or the function of expressed α4β2 and α7 nAChRs.

Conclusion:

Our results support the potential of L-theanine as a promising candidate for treating nicotine dependence.

Keywords: L-theanine, nicotine, withdrawal, Conditioned Place Preference, mice, nicotinic receptors

1. Introduction

Smoking is of the leading source of preventable disease and premature death worldwide. Approximately 550,000 people in the United States die prematurely from smoking-related diseases every year (Carter et al., 2015; Doll et al., 2004). Tobacco use involves in several life-threatening illnesses such as pulmonary and cardiovascular diseases, cancer and other disorders (CDC, 2009; U.S. DHSS, 2014). Nicotine, a natural alkaloid of tobacco, is responsible for the initiation and maintenance of smoking addiction. Nicotine induces positive reinforcing and rewarding responses and upon its cessation a withdrawal syndrome (George et al., 2007; Kenny and Markou, 2001; Le Foll and Goldberg, 2009). The current pharmacotherapy of smoking dependence, which include nicotine replacement therapy, bupropion, and varenicline, is of limited success. Indeed, the rate of success for 1-year abstinence is only about 20–25% with any present pharmacotherapies (George and O’Malley, 2004; Aubin et al, 2014) and therefore it is critical to develop more effective treatments.

L-theanine, 2-amino-4-(ethylcarbamoyl) butyric acid, an amino acid presents in green tea (Camellia sinensis) and mushrooms (Boletus badius) that crosses the blood brain barrier (Yokogoshi et al., 1998), is consumed due to its favorable taste as well as its ability to induce a relaxing effect on the body. In addition, L-theanine was shown to produces anxiolytic properties in humans (Kimura et al., 2007; White et al., 2016). Recently, L-theanine was shown to attenuate nicotine conditioned reward in the mouse conditioned place preference (CPP) test at a high range of doses (Di et al., 2012). These results suggest that L-theanine may suppress withdrawal signs associated with nicotine dependence.

We investigated whether L-theanine attenuates nicotine withdrawal signs in mice as well as nicotine rewarding properties using the CPP test. We also evaluated the effect of L-theanine administration on acute nicotine pharmacological responses in mice and its effects on expressed nicotinic receptors. The results of this study will enhance our knowledge of the therapeutic potential of L-theanine for nicotine dependence.

2. Materials and Methods

2.1. Animals

Male ICR mice (8 weeks upon arrival; Harlan Laboratories, Indianapolis, IN) used as subjects. Four mice were housed per cage with ad libitum access to food and water. The ICR mice were housed in a vivarium, featuring a 12-h light cycle and a 21 °C humidity- and temperature-controlled environment. The facility was approved by the Association for Assessment and Accreditation of Laboratory Animal Care. All the studies were performed during the animals’ light cycle and were approved by the Institutional Animal Care and Use Committee of Virginia Commonwealth University and followed the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals.

2.2. Drugs

(−)-Nicotine hydrogen tartrate [(−)-1-methyl-2-(3-pyridyl) pyrrolidine (+)-bitartrate], mecamylamine HCl and L-theanine were obtained from Sigma-Aldrich Inc. (St. Louis, MO, USA). The drugs were dissolved in physiological saline and solutions were administered subcutaneously (s.c.) at 10 ml/kg. Each dose is expressed as the free base of the drug. The doses and pretreatment times of L-theanine used in the study were based on previous reports (Wise et al., 2012). [3H]epibatidine (56.3 Ci/mmol) and [3H] tetracaine (30 Ci/mmol) were purchased from PerkinElmer Life Sciences and Sibtech, Inc., respectively.

2.3. Withdrawal Studies

Mice received nicotine (24 mg/kg) or saline for 14 days, delivered by s.c. osmotic mini pumps (model 2000; Alzet Corporation, Cupertino, CA) as described by our previous report (Jackson et al., 2008). The mini pumps were implanted under isoflurane anesthesia. Nicotine concentration enclosed in each mini pump was calculated taking into account mice weight and mini pump flow rate. On the morning of day 15, mice were pretreated with vehicle or L-theanine (3, 6, 12 or 24 mg/kg, s.c.) 20 min prior to mecamylamine (2 mg/kg, s.c.) administration (n=8/group). Mecamylamine is a nonselective nicotinic antagonist used to precipitate nicotine withdrawal.

Ten minutes after the mecamylamine administration, Light-Dark Box (LDB) test was performed, followed by somatic signs observation and then hyperalgesia in the hot-plate test (Jackson et al., 2008). This order of testing was shown previously by our group, to reduce within-group variability and produced the most consistent results (Jackson et al., 2008). All studies were performed by an observer blinded to experimental treatment.

The LDB testing was conducted as previously reported (Wilkerson et al., 2016). The LDB equipment is formed of a small dark chamber (36 × 10 × 34 cm) linked to a larger light chamber (36 × 21 × 34 cm) via an entryway (6 × 6 cm). Mice were placed in the test room 30 min prior to the assay to habituate. Each mouse began the test in the light chamber and animals were having free access to explore the entire apparatus for 5 min. Using video monitoring and ANY-MAZE software (Stoelting Co., Wood Dale, IL), the mice’s movements were recorded and categorized. The number of entries into the light chamber, the number of transitions between the two chambers, and the total time spent in the light chambered were reported.

Somatic signs which include paw and body tremors, head shakes, backing, jumps, curls, and ptosis were observed for 30 minutes for each individual mouse. During the observation period, mice were placed in clear activity cages without bedding. The average number of total somatic signs was determined for each treatment group.

Immediately following somatic signs session, mice were evaluated for hyperalgesia using the hot plate test. Mice were placed in a 10-cm wide glass cylinder on a 52°C hot plate (Thermojust Apparatus, Richmond, VA). The latency to reaction time (jumping or paw licking) was noted.

2.4. Conditioned place preference (CPP) studies

CPP test (unbiased design) was performed as previously explained (Jackson et al., 2019). In brief, the apparatus of CPP is formed of three chambers in a row: a smaller gray chamber bounded by a white and black chamber (ENV3013; Med Associates, St Albans, VT). The external white and black chambers are larger (20×20×20 cm each) and differ in floor texture. The white chamber floor is composed of white mesh. The black chamber floor is composed of black rods. The passages connecting the white and black chambers to the gray chamber have smooth PVC flooring. Partitions can be placed to block the mouse from moving between the chambers.

On day 1, the ICR animals were put in the middle gray chamber for a 5 min habituation and then allowed free access to the white and black chambers for period of 15 min. The time spent in each chamber was recorded and each mouse’s baseline chamber preference for either the white or black chamber was found. Experimental groups were divided with mice of approximately equal bias in baseline chamber preference.

Twenty-minute conditioning sessions were performed by researcher twice a day (3 days). All sessions were 4 hr apart and conducted by the same investigator. In conditioning sessions, a subject was confined to either the white or black box and administered either nicotine (0.5 mg/kg, s.c.) or saline. The treatment group injected with one dose of nicotine a day in the drug paired chamber. For the other daily conditioning shift, the drug cohort received vehicle (saline) in the non-drug paired box. Administrations of treatment and vehicle were counterbalanced to make certain some mice had the unconditioned stimulus in the morning and other cohort received it in the afternoon session (pavlovian conditioning). Mice received L-theanine pretreatment (1, 3, or 6 mg/kg, s.c.) or vehicle pretreatment 20 min before nicotine (n=10/group). The control groups obtained saline in the large box in the morning and saline in the other large box in the afternoon session.

On test day (day 5), each mouse was placed in the CPP apparatus with no partitions for 15 min in a drug free state. The time spent in each chamber was recorded. The preference score was determined by calculating the difference in time spent in the drug paired chamber on the test day versus the time in drug paired chamber on the baseline day.

2.5. Acute Nicotine Studies

In these experiments, we studied the effects of the two highest doses of L-theanine (12 and 24 mg/kg, s.c.; 60 min pretreatment) used in the dependence studies on acute responses of nicotine. Two responses to nicotine at a low (0.5 mg/kg, s.c.) and high dose (2.5 mg/kg, s.c.) were measured after administration: antinociception using the tail-flick test and changes in body temperature. Separate groups of mice were used for each nicotine and L-theanine doses.

Antinociception.

Antinociception was measured using the tail-flick assay adapted from D’Amour and Smith (1941). A radiant heat source was applied to the upper portion of the tails of lightly restrained mice. The response time of the mouse, visualized by a tail flick, was recorded. The heat source automatically turns off after 10 s, minimizing tissue damage. A baseline response (typically 2–4 s) was determined for each mouse before treatment, which acted as the control. Mice were then reassessed 5 min after nicotine injection. The antinociceptive response was expressed as percentage of maximum possible effect (%MPE), calculated as %MPE = [(test − control)/(10 − control)] × 100. Increased latency is indicative of antinociception.

Body temperature.

To measure the mice’s rectal temperature, the researcher utilized a thermistor probe (inserted 24 mm) and a digital thermometer (YSI Inc., Yellow Springs, OH). Readings were taken immediately prior to and 30 min following nicotine administration. The change in rectal temperature pre- and post-treatment was calculated for each mouse. Throughout the study, the ambient temperature of the experiment room varied from 21–24°C.

2.6. Radioligand binding analyses:

Filtration-based assay previously described (Jayakar, Ang et al. 2017, Appiani, Pallavicini et al. 2022) was used to determine the effect of L-theanine on the reversible binding of [3H]epibatidine and [3H]tetracaine to membrane isolated from HEK-cells stably expressing the human α4β2 nAChR (HEK-hα4β2). Briefly, HEK-hα4β2 membranes (10–25 μg protein) were incubated at room temperature for 2 h with [3H]epibatidine (2.5 nM) or [3H]tetracaine (50 nM) in the absence of other ligands (total binding) or the presence of 1 mM L-theanine. Nonspecific bindings of [3H]epibatidine and [3H]tetracaine were determined in the presence of 100 μM cytisine or 1 mM imipramine, respectively. Receptor-bound and free 3H were separated by rapid filtration using polyethyleneimine-treated glass microfiber filters and receptor-bound 3H (filters) were quantified by liquid scintillation counting.

2.7. In vitro electrophysiological recordings:

Two-electrode voltage clamp whole current recoding from Xenopus laevis oocytes heterologously expressing nAChRs was used to study the effects of L-theanine on nAChRs function as previously described ((Wang, Deba et al. 2017, Deba, Munoz et al. 2021). Briefly, oocytes were surgically harvested from Xenopus laevis following animal use protocol approved by the University of Texas at Tyler Institutional Animal Care and Use Committee and processed for injection with cRNA transcripts to express the α4β2 nAChR or human α7 nAChR. Once nAChR expression is confirmed, L-theanine (1–1000 μM) was applied alone or in combination with the agonist ACh (10 or 1000 μM) for 10s separated by 3–5 min wash intervals. Peak current responses were normalized to current response to ACh alone applied within same recording run. Then normalized current responses from N oocytes are pooled and shown in graph as average ± SD.

2.8. Statistical analysis

The data were analyzed using mixed-factor analysis of variance (ANOVA), performed by the GraphPad Prism software, version 9 (GraphPad Software, Inc., La Jolla, CA). The results are expressed as the mean ± S.E.M. Two-way ANOVA were used to determine the effect of L-theanine in the withdrawal assays and one-way ANOVA in the CPP test as well as in the acute nicotine studies. Prior to statistical analysis, the data were first assessed to determine if the assumptions of a one-way ANOVA were met. Normal distribution was assessed using the F-test. Equal variation was assessed using the Brown-Forsythe test and Bartlett’s test for one-way ANOVA or Levene’s test for two-way ANOVA. All data passed these tests. Significant overall ANOVAs are followed by Tukey’s post hoc correction when appropriate. All differences were considered significant at p < 0.05. Binding and in vitro electrophysiological data analyses were plotted using GraphPad Prism software version 9 (GraphPad Software, Inc., San Diego, CA, USA).

3. Results

3.6. Impact of L-theanine on nicotine withdrawal

Anxiety-related behaviors, somatic signs and hyperalgesia were assessed in mice who underwent mecamylamine-induced nicotine withdrawal 15 days after s.c. minipump implantation. Mice were pretreated with either L-theanine or vehicle 60 min prior to mecamylamine administration. As shown in Fig. 1A, mecamylamine injection significantly increased number of somatic signs in nicotine-treated group comparison to their saline treated counterparts [F(pretreatment) (1, 70) = 248.7; P<0.0001]. In addition, saline-infused mice injected with vehicle showed no significant withdrawal signs. Interestingly, administration of L-theanine (3, 6, 12 and 24 mg/kg, s.c.) dose-dependently decreased nicotine withdrawal somatic signs [F(treatment) (4, 70) = 29.58; P<0.0001; F(interaction) (4, 70) = 27.06; P<0.0001; Fig. 1A]. Furthermore, nicotine-treated group showed a significantly higher hyperalgesia comparing to saline-infused mice [F(pretreatment) (1, 56) = 58.26; P<0.0001; Fig. 1B]. Administration of 12mg/kg dose of L-theanine significantly reversed nicotine-induced hyperalgesia in the hot plate test [F(treatment) (3, 56) = 1.819; P=0.1541; F(interaction) (3, 56) = 3.867; P=0.0139; Fig. 1B]. Similarly, administration of 12 mg/kg of L-theanine significantly reduced the anxiety-like response of nicotine-treated group compared with saline controls in LDB [F(treatment) (3, 56) = 6.572; P=0.0007; Fig. 1C]. There was a significant interaction between L-theanine and nicotine [F(3, 56) = 2.719; P=0.0531; Fig. 1C]. No significant difference was found in the number of transitions between nicotine and L-theanine treated groups (Table 1).

Figure 1. Effect of L-theanine on nicotine precipitated withdrawal in mice.

Figure 1.

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Mice were chronically infused with saline or nicotine (24 mg/kg/day) for 14 days. On the morning of day 15, mice were pretreated with vehicle, L-theanine (3, 6, 12 and 24 mg/kg, s.c.; 20 min prior) before challenge with mecamylamine (2 mg/kg, s.c.) to precipitate withdrawal. A) Effects of L-theanine on somatic signs, B) Hyperalgesia in the hot plate test and C) Light Dark Boxes. Each point represents the mean ± S.E.M. of 8 mice per group. * Denotes p<0.05 from vehicle-Saline. # Denotes p<0.05 from vehicle-nicotine. Open box = Saline minipumps, closed boxes = nicotine minipumps.

Table 1. L-theanine did not influence the average number of transitions in the Light-Dark Box (LDB) test.

Mice undergoing nicotine withdrawal received L-theanine (3, 6 and12 mg/kg; s.c.) or vehicle. The average number of transitions across chambers were recorded in the LDB test. The numbers are presented as the mean ± SEM of the total transitions (n=8/group). MP = Minipumps.

Treatment Average number of transitions ±SEM
Saline MP-vehicle 10.25± 0.98
Saline MP-L-theanine (3mg/kg) 11.25± 1.45
Saline MP-L-theanine (6mg/kg) 10.75± 1.69
Saline MP-L-theanine (12mg/kg) 11.50± 1.88
Nicotine MP-vehicle 10.88± 0.95
Nicotine MP-L-theanine (3mg/kg) 10.38± 1.18
Nicotine MP-L-theanine (6mg/kg) 10.75± 1.22
Nicotine MP-L-theanine (12mg/kg) 11.88± 1.53
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3.7. L-theanine attenuated the nicotine induced conditioned place preference

Conditioning with either saline or nicotine (0.5 mg/kg) was performed for 3 days showed significant preference in nicotine-conditioned mice pre-treated with vehicle [F (5, 54) = 19.49; p < 0.0001). Post hoc analysis revealed that pretreatment with the lower doses of L-theanine, 1 and 3 mg/kg, did not significantly alter nicotine CPP (p>0.05), but a higher dose of 6 mg/kg significantly reduced nicotine conditioned place preference (p<0.05) (Fig. 2). L-theanine at 6 mg/kg on its own did not produce a preference or aversion in mice.

Figure 2. L-theanine attenuated nicotine preference in the CPP test.

Figure 2.

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Mice were conditioned with either saline or nicotine (0.5 mg/kg s.c.) for 3 days. Nicotine treated mice showed significant conditioned place preference. L-theanine at 6mg/kg significantly reduced the nicotine preference. * Denotes p<0.05 from vehicle-saline. # Denotes p<0.05 from vehicle-nicotine. Each point represents the mean ± SEM of 10 mice per group. Open box = Saline group, closed boxes = nicotine group.

3.8. L-theanine did not alter acute nicotinic pharmacological responses

To determine L-theanine’s effect on nicotine in vivo pharmacological sensitivity, we measured the impact of L-theanine on behavioral nicotine responses after acute dosing at a low dose (0.5 mg/kg) and a high dose (2.5 mg/kg) of the drug in mice. Different cohorts of mice were pretreated with L-theanine, either 12 or 24 mg/kg, 60 min prior to a single acute injection of nicotine (either 0.5 or 2.5mg/kg) or saline and tested in the following pharmacological responses: body temperature and antinociception effect (tail-flick test). According to the Ordinary One-way ANOVA, mice injected with a low dose of nicotine revealed significant hypothermic [F(5, 42) = 10.27; p<0.05) and antinociceptive [F(5, 42) = 5.739; p<0.05] effects (Fig. 3A and 3B). However, injection of L-theanine did not significantly change the nicotine induced-antinociception and hypothermic response (p>0.05) (Fig. 3A and 3B). Similarly, the high dose of nicotine significantly decreased body temperature (hypothermia) [F(5, 42) = 88.99; p<0.05] and induced antinociception in comparison to the saline injected cohort of mice (P<0.05) (Fig. 3C and 3D). However, exposure to L-theanine did not block the nicotine-induced hypothermia (P> 0.05) (Fig. 3C) and antinociception [F(5, 42) = 81.71; p<0.05] (Fig. 3D). In addition, L-theanine did not induce any changes (P> 0.05) on its own in these assays. These findings suggest that L-theanine did not alter the initial pharmacological responses of nicotine.

Figure 3. L-theanine has no effect on acute nicotinic pharmacological responses.

Figure 3.

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Effects of L-theanine administration on A) Body temperature, B) Antinociception after nicotine (0.5 mg/kg s.c.) or saline administration (s.c.); C) Body temperature, D) Antinociception after nicotine (2.5 mg/kg s.c.) or saline administration (s.c.). L-theanine (12 and 24 mg/kg, s.c.) was injected 60 min prior to a single acute injection of nicotine or saline. L-theanine at both doses did not affect nicotine-induced hypothermia and antinociception in mice. * Denotes p<0.05 from vehicle-saline. Each point represents the mean ± S.E.M. of 8 mice per group. Open box = Saline group, closed boxes = nicotine group.

3.9. Interaction of L-theanine with α4β2 and α7 nAChRs:

To determine if L-theanine binds to the agonist-binding site or the ion channel of the α4β2 nAChR subtype, we examined the ability of L-theanine to displace the bindings [3H]epibatidine (nAChR agonist; (Xiao and Kellar 2004)) and [3H]tetracaine (noncompetitive antagonist; (Middleton, Strnad et al. 1999)) to HEK-hα4β2 membranes (Figure 4). L-theanine at 1 mM did not displace [3H]epibatidine or [3H]tetracaine indicating that L-theanine does not bind to the agonist binding site or the ion channel of α4β2 nAChR. Figure 4 also shows the effect of the nAChR agonist cytisine on [3H]epibatidine binding and the effect of nAChR noncompetitive antagonist imipramine on [3H]tetracaine binding for comparison.

Figure 4.

Figure 4.

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[3H]epibatidine (A) and [3H]tetracaine (B) binding to HEK-hα4β2 in the absence of any ligand, presence of 1 mM L-theanine (A and B), presence of cytisine (B), or presence of imipramine (B).

In parallel, we examined the direct effect of L-theanine on the α4β2 and α7 nAChRs current as well as the effect of L-theanine on agonist-induced current responses of these receptors (Figure 5). When applied to oocytes expressing α4β2 or α7 nAChRs, L-theanine up to 1 mM did not induce current activity indicating that L-theanine does not directly activate (i.e. has no agonistic effect) α4β2 or α7 nAChRs (data not shown). When L-theanine (1–1000 μM) co-applied with sub-maximal (~EC10 and ~EC50) and maximal (1 mM) ACh concentrations, it did not significantly enhance or inhibit ACh-induced current of α4β2 or α7 nAChRs. When L-theanine was co-applied with 10 μM ACh to α4β2 nAChR, there was no enhancement of AC-induced currents and the maximum inhibition observed was 76±18% of control at 1000 μM L-theanine and when co-applied with 100 μM ACh inhibition was <12% of control at any L-theanine concentration tested (Table 2). Altogether, these data support a lack of direct interaction between L-theanine and α4β2 and α7 nAChRs.

Figure 5: L-theanine did not inhibit ACh-induced current responses of hα4β2 or hα7 nAChR.

Figure 5:

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Current responses to 1000 μM ACh alone or in the presence of 1000 μM L-theanine were recorded from Xenopus oocytes expressing hα4β2 or hα7 nAChR. For each application, peak current amplitude was quantified and normalized to peak current amplitude elicited by 1000 μM ACh alone within the same recording run. Replicas from the same oocyte were averaged (shown as circles) and data (average ± SD) from N oocytes are shown as bar graph.

Table 2.

Current responses to 10 pM or 100 pM ACh alone or in the presence of increasing concentrations of L-theanine were recorded from oocytes expressing ha4^2 nAChRs. For each application peak current amplitude was quantified and normalized to peak current amplitude elicited by 10 pM or 100 pM ACh alone within the same recording run. Replicas from the same oocyte were averaged and for each L-theanine concentration (average ± SD) of data from N oocytes were listed.

L-theanine concentration (μM) I(10uM ACh + L-theanine)/I(10uM ACh)
Ave ± SD (N)		 I(100uM ACh + L-theanine)/I(100uM ACh)
Ave ± SD (N)
1 99±3 (6) 113±4 (3)
3 97±5 (4) 100±4 (3)
10 101±4 (3) 87±3 (3)
30 98±2 (3) 114±4 (3)
100 92±4 (4) 92±8 (3)
300 78±11 (3) 89±16 (3)
1000 76±18 (2) 94±8 (4)
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4. Discussion

The present report is the first to preclinically evaluate the amino acid L-theanine’s effect on nicotine withdrawal. We demonstrated that systemic administration of L-theanine decreased nicotine withdrawal affective and somatic manifestations in ICR male mice. In addition, our results revealed that L-theanine blocked the development of nicotine preference in the CPP test. However, L-theanine did not alter acute nicotine responses (body temperature and antinociception effect) in mice nor the binding and function of expressed α4β2 and α7 nAChRs.

Our novel findings show that L-theanine reduced both physical (somatic signs and hyperalgesia) and affective (anxiety-related behavior) nicotine withdrawal signs in mice. L-theanine dose-dependently reduced nicotine somatic signs at all doses tested. However, L-theanine significantly attenuated hyperalgesia and anxiety-like behaviors withdrawal signs only at the highest dose tested of 12 mg/kg. Importantly, L-theanine at this dose did not elicit an effect on its own in the LDB. This is consistent with a recent study that reported anxiolytic-like effects at higher doses of L-theanine (16 and 24 mg/kg) in the elevated plus maze test in mice (Wise et al., 2012). As L-theanine is known to produce general relaxing and anxiolytic effects in humans, we cannot fully conclude that this decrease in anxiety-like behavior in mice is specific to nicotine (Haskell et al., 2008; Lu et al., 2004). The difference in L-theanine potency to block the various nicotine withdrawal signs may be explained by the fact that these signs are mediated by different nicotinic acetylcholine receptor subtypes and neuronal pathways (Jackson et al., 2009; Jackson et al., 2008).

Our findings with nicotine physical dependence is further supported by prior work examining L-theanine’s effect on morphine withdrawal (Wise et al., 2012). In the aforementioned report, L-theanine decreased observed spontaneous withdrawal signs in morphine-dependent rhesus monkeys in a dose-dependent fashion. Taken together with our current observations, L-theanine’s effects are not specific to morphine but also extend to nicotine. In addition, our data on nicotine withdrawal induced-hyperalgesia is consistent with a recent report where L-theanine was shown to alleviate thermal hyperalgesia in a rat nerve injury model (Chen et al., 2022).

Our results in nicotine CPP are consistent with an earlier work that showed L-theanine reduced nicotine conditioned reward in the CPP test in mice (Di et al., 2012). However, the L-theanine dose reported in Di et al. was very high (500 mg/kg, s.c.) compared to the small dose range of L-theanine used in our study (1–6 mg/kg, s.c.). Di et al. additionally found L-theanine blocked nicotine’s increase in tyrosine hydroxylase and dopamine levels in the mouse midbrain, suggest a potential mechanism of action for L-theanine’s effect on nicotine reward. The CPP data needs to be interpreted with caution since it is possible that L-theanine administration caused an impairment on Pavlovian learning which is a process on which CPP relies. However, human and animal reports suggest that L-theanine does in fact enhances cognition and memory (Williams et al., 2019).

While L-theanine reduced nicotine withdrawal signs and nicotine CPP, it did not alter the acute pharmacological responses of nicotine. We tested the effect of L-theanine on the acute response to two doses of nicotine. L-theanine did not elicit any significant changes in nicotine induced-antinociception and hypothermic response, suggesting L-theanine does not directly block nicotine preference in the CPP test.

As this is a preliminary animal study, our findings do not provide a clear mechanism by which L-theanine blocks nicotine withdrawal and reward in the mouse. Consistent with our in vivo data with acute responses of nicotine, our in vitro results showed that L-theanine did not directly modulate the primary pharmacological target of nicotine, in particular α4β2 nAChR subtypes. Alternatively, L-theanine may regulate the expression and levels of brain nAChRs after chronic nicotine exposure, leading to a change in nicotine withdrawal symptoms. Several nAChRs subunits were shown to mediate the affective signs of nicotine withdrawal. For example, the α6 nAChR subunit is involved in nicotine withdrawal induced-anxiety and anhedonia-like behaviors (Alkhlaif et al., 2017; Jackson et al., 2009). The somatic signs associated with nicotine withdrawal syndrome are mediated by α3 (Jackson et al., 2013), α5 (K J Jackson et al., 2008; Salas et al., 2009), α2 (Salas et al., 2009), β4 (Jackson et al., 2013; Stoker et al., 2012) and α7 nicotinic subunits (Grabus et al., 2005; Jackson et al., 2008). In addition, L-theanine pretreatment at very high doses reduced nicotine-induced upregulation of various nAChR subunits in the some brain regions implicated in nicotine dependence: α4 and β2 in the prefrontal cortex and α4, β2, and α7 in the VTA and the nucleus accumbens (Di et al., 2012). It is possible therefore that L-theanine’s decrease of nAChRs upregulation mediate the decrease in nicotine withdrawal observed in our study. It has been suggested that nAChR upregulation may contribute to nicotine withdrawal as receptor density increase is positively correlated to withdrawal signs and craving (Cosgrove et al., 2009; Gould et al., 2012; Wilkinson et al., 2013).

Other possible mechanisms could mediate the effects of L-theanine on nicotine dependence, in particular the neuronal glutamate system. Nicotine’s conditioned reward effect is crucially mediated by glutamate signaling (Wang et al., 2010). The blockage of N-methyl-D-aspartate (NMDA) receptors attenuates nicotine-induced CPP (McGeehan and Olive, 2003; Wang et al., 2010; Yararbas et al., 2010). L-theanine has been reported to have an antagonistic effect on NMDA receptors (Yokogoshi et al., 1998), potentially explaining L-theanine’s effects on nicotine CPP. In addition, L-theanine increases CNS levels of GABA, dopamine, and serotonin, as well as inhibits glutamate receptors (Kimura and Murata, 1971; Yokogoshi et al., 1998). Moreover, studies indicate that L-theanine increases BDNF levels (Takeda et al., 2011; Wakabayashi et al., 2012). L-theanine may produce its actions on nicotine withdrawal and CPP via its effects on these neurotransmitters.

While the current results suggest a possible modulatory role of L-theanine on important aspects of nicotine dependence, some limitations should be noted concerning the potential translation of our findings. First, we evaluated the effect of L-theanine on various nicotinic behaviors in male mice. Secondly, we assessed the impact of L-theanine on precipitated nicotine withdrawal and not spontaneous withdrawal, a more relevant approach. Finally, L-theanine was given in subcutaneous injections instead of oral administration in animals. While the blockade of nicotine preference by L-theanine is suggestive of a decrease in the rewarding properties of nicotine, we have not tested L-theanine on nicotine intake using nicotine self-administration animal models to assess its effectiveness on drug-seeking behavior and reinstatement. Lastly, we did not measure the plasma and brain levels of L-theanine in our study. Earlier studies have indicated L-theanine reaches the brain following systemic administration in rodents without undergoing significant metabolic changes (Yokogoshi et al., 1998).

Taken all together, our findings suggest this relatively safe dietary supplement, L-theanine, may be useful in the alleviation of the nicotine withdrawal syndrome in humans and possibly decrease the positive reinforcement effect of nicotine.

Highlights.

  • L-theanine has anxiolytic effects in mice.

  • L-theanine induces reduction in nicotine withdrawal sighs.

  • L-theanine attenuates nicotine conditioned place preference.

  • L-theanine, may be useful therapy of the nicotine withdrawal syndrome in humans and possibly decreases the nicotine reward effects too.

Acknowledgements:

This study was supported by NIH grant R01 DA032246 (MID) and UT Tyler-Office of Research and Scholarship-Faculty Research Support (AKH).

Abbreviations:

nAChR

nicotine acetylcholine receptor

LDB

light dark box

CPP

Conditioned place preference

Footnotes

Conflicts of interest: None declared.

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