Levo-Tetrahydroberberrubine Produces Anxiolytic-Like Effects in Mice through the 5-HT1A Receptor

Tetrahydroprotoberberines (THPBs) are isoquinoline alkaloids isolated from the Chinese herb Corydalis yanhusuo. In the present study, we performed competitive binding assays to examine the binding of l-THBr to neurotransmitter receptors known to be involved in sedation, hypnosis and anxiety. Our results show that l-THBr does not interact with GABAergic receptors but has binding affinities for dopamine and serotonin receptors. In addition, cAMP and [35S]GTPγS assays were used to determine the agonist or antagonist properties of l-THBr at dopamine (D1, D2) or serotonin (5-HT) receptors. Our results show that l-THBr displays D1 and D2 antagonist and 5-HT1A agonist properties. Moreover, l-THBr-treated rodents exhibit anxiolytic-like effects in the light/dark box and elevated plus-maze tests, and the anxiolytic effect of l-THBr can be reduced by WAY-100635, a selective 5-HT1A receptor antagonist. Our results suggest that l-THBr may produce potent anxiolytic-like effects mainly through serotonin receptors.


Introduction
Anxiety is a common mental state provoked in anticipation of a threat or potential threat, which may become an illness when excessive or inappropriate [1,2]. The major physical and mental symptoms of anxiety include racing thoughts, nervousness, tremor, insomnia, emotional discomfort and agitation [3,4]. As one of the most common psychiatric illnesses, anxiety disorders cause a prominent health care problem worldwide.
For over a century, researchers have searched for effective and safe agents to treat anxiety disorders. Benzodiazepines have been the mainstay of treatment since chlordiazepoxide was introduced in 1960 [5]. However, their therapeutic efficacy is limited due to unwanted side effects such as sedation, muscle relaxation, retrograde amnesia [6,7] and dependency liability [8]. Another class of drugs, partial agonists of the serotonergic 5-HT 1A receptor, such as buspirone, gepirone, and ipsapirone, was identified as valuable for improving the clinical management of anxiety [9], but their therapeutic effects are delayed for 1-3 weeks [10]. Therefore, a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 there is a demand for robust anxiolytic compounds that have fewer side effects and a more immediate onset of action.
Tetrahydroprotoberberines (THPBs) are isoquinoline alkaloids isolated from the Chinese herb Corydalis yanhusuo. l-Tetrahydropalmatine (l-THP), the main active ingredient of C. yanhusuo, has been used for more than 40 years in China as a treatment for chronic pain and anxious insomnia [11,12]. l-THP displays D 1 and D 2 antagonist properties and shows antiaddictive effects in animal models [13][14][15][16]. In addition, l-stepholidine (l-SPD), another derivative of tetrahydroprotoberberines, displays D 1 agonist and D 2 antagonist effects [17]. l-SPD has attracted much attention for its potential efficacy as a schizophrenia treatment [18][19][20]. However, l-SPD's poor bioavailability and high industrial production cost limits its use [21]. Therefore, a new derivative of THPBs, levo-tetrahydroberberrubine (l-THBr) (Fig 1) was synthesized. In the present study, we examined the binding features of l-THBr to neurotransmitter receptors using competitive binding assays to address possible interactions with these receptors. Moreover, we characterized the functional activity of l-THBr at cloned D 1 and D 2 dopamine receptors and rat hippocampal 5-HT 1A serotonin receptors. In addition, the anxiolytic-like effects of l-THBr in two experimental animal models of anxiety were evaluated.

Animals
The experimental procedures were approved by the Beijing Institute of Basic Medical Science Institutional Committee on Animal Care and Use, and all efforts were made to minimize animal suffering and reduce the number of animals used for experiments. Male Sprague-Dawley (SD) rats and male CD-1 ICR mice (body mass of 18-22 grams) were purchased from Vitalriver Experimental Animal Center (Beijing, China). All animals were maintained under standard laboratory conditions and kept in temperature-and humidity-controlled rooms (21-22˚C, 50%-60% humidity) on a 12 hour light-dark cycle (lights on from 7:00 am to 7:00 pm). All mice were used only once, and all behavioral experiments were performed between 8:00 and 12:00 am.

Radio-ligand binding assay
To determine the possible targets of l-THBr action, the binding affinities of l-THBr to neurotransmitter receptors known to be involved in sedation, hypnosis and anxiety were investigated. Radio-ligand binding assays were performed by Caliper Lifescience (Hopkinton, MA, USA). The screening was carried out at a concentration of 10 μM l-THBr to test its ability to inhibit the binding of radioligands to their corresponding receptors. The results were expressed as percentage of inhibition of labeled ligand binding to individual receptors. Significant binding activity was defined as !50%.
cAMP assay for binding properties at D 1 receptor Dopamine receptors can be categorized into two classes: Gαs protein coupled receptors (D 1 and D 5 ), or Gαi protein coupled receptors (D 2 , D 3 , and D 4 ). Activation of D 1 receptors can excite adenylate cyclase activity and increase cyclic adenosine monophosphate (cAMP). cAMP assays were used to determine the agonist or antagonist properties of l-THBr at the dopamine D 1 receptor.
CHO K1 cells stably expressing D 1 receptors were purchased from Genscript (Catalogue Number: M00247, Gene Number NM_000794). The cells were seeded in Ham's F12 containing 10% fetal bovine serum and 200 μg/ml zeocin. On the day of the assay, 5 μl of cell suspension (3000 cells) was seeded on a 384-well plate. The assay was performed according to the manufacturer's instructions. To test for agonist effects at the D1 receptor, the compounds (a known D 1 agonist SCH38393 or l-THBr) were added from stocks two-fold more concentrated than the final concentration. In another experiment to test for antagonist effects, the known D 1 receptor antagonist SCH23390 or l-THBr was added to the system in the presence of the D 1 receptor agonist SCH38393 (10 μM). After incubation (30 min at room temperature), 10 μL of HTRF reagents (cAMP-XL665 and anti-cAMP cryptate) were added. The signal was quantified after one hour of incubation at room temperature. The fluorescence intensity ratio (A 665nm / A 620nm x10 4 ) was calculated.
[ 35 S]GTPγS assay for binding properties at D 2 and 5-HT 1A receptors D 2 -D 4 dopamine receptors and 5-HT 1A serotonin receptors are Gαi-coupled receptors that mediate inhibitory neurotransmission. We conducted [ 35 S]GTPγS assays to determine the agonist or antagonist properties of l-THBr at D 2 dopamine receptors or 5-HT 1A serotonin receptors. HEK293 cells stably expressing D 2 receptors were provided by the Beijing Institute of Pharmacology and Toxicology (Beijing, China). The membrane preparation of D 2 -expressing HEK293 cells and rat hippocampal tissues highly expressing 5-HT 1A receptors were prepared as previously described [22][23]. Briefly, 10 SD rats were anesthetized and decapitated, and the hippocampi were quickly dissected and stored at -80˚C until use. D 2 receptor-expressing HEK293 cells and the hippocampal tissue were each homogenized at 4˚C in 50 mM Tris-HCl buffer (pH 7.4). The homogenates were centrifuged at 2500 × g for 6 min, and the supernatant was further centrifuged for 20 min at 40000 × g. Membranes were re-suspended in Tris-HCl buffer and stored at -80˚C until use.

Locomotor activity test
The spontaneous activity video analysis system consists of 8 sound-attenuated chambers (40 cm × 40 cm × 65 cm) with a built-in infrared camera. (JL Behave, Shanghai Ji-Liang Software Technology Co., Ltd). During the behavioral tests, the experimenter was outside the testing room, and the chambers were cleaned between successive runs. Forty male CD-1 ICR mice were allowed to acclimate for three days in home cages and were handled for another three days to minimize stress after arrival in the animal facility. On the following day, after habituation to the activity chambers for 60 min, mice were administered vehicle, diazepam (DZP, 2 mg/kg, i.p.) or l-THBr (1, 5, or 10 mg/kg, i.p.) and immediately placed into the test chambers to record their locomotor activity for 60 min.

Light-dark box test
The light-dark box test is a sensitive model to detect activity in disorders related to anxiety, based on the innate aversion of rodents to brightly lit areas and on their spontaneous exploratory behavior in response to a novel environment [24]. The light-dark transition box is a polypropylene animal cage (44 cm × 21 cm × 21 cm), which is divided into two compartments, a light box (illuminated by a 60 W light source with 1000 lx light intensity) and a dark box. Forty-eight male CD-1 ICR mice were placed in the light box 30 min after l-THBr or DZP injection and allowed to move freely to both boxes for 5 min. The number of transitions between the two boxes were recorded by a video camera.

Elevated plus-maze test
The elevated plus-maze test is widely used for the screening and evaluation of anxiolytic drugs [25][26]. The apparatus consists of two open arms (30 cm × 5 cm) and two enclosed arms (30 cm × 5 cm × 15 cm), which is elevated 45 cm above the ground. The entire maze was made of clear Plexiglas and illuminated by four 30 W white lights with 300 lx light intensity arranged as a cross 100 cm above the maze. 48 male CD-1 ICR mice were randomly divided into either the vehicle group, one of three doses of l-THBr groups, or the diazepam group. In a separate experiment, WAY100635 (a 5-HT 1A antagonist) was used to test whether the anxiolytic-like activity of l-THBr is mediated by the activation of the 5-HT 1A receptor. Forty male CD-1 ICR mice were randomly divided into one of four groups: vehicle, WAY100635 (3 mg/kg, i.p.), l-THBr (5 mg/kg, i.p.) or WAY100635 (3 mg/kg, i.p.) + l-THBr (5 mg/kg, i.p.). Mice were administered vehicle or WAY100635 followed by vehicle or l-THBr (5 mg/kg) injections 15 min later. The test was performed 30 min after the administration of l-THBr or vehicle.

Statistical analysis
All data sets were initially checked for normality and homogeneity of variance. The data were expressed as the mean ± S.E.M and assessed using one-way ANOVA followed by Bonferroni post hoc comparisons. A 2 x 2 factorial ANOVA was used to determine interaction effects for WAY100635 and l-THBr. P < 0.05 was defined as a statistically significant difference.

Binding affinity of l-THBr to neurotransmitter receptors
The in vitro receptor competitive binding data illustrate that l-THBr at a concentration of 10 μ M has a high binding affinity for D 1 , D 2 , and D 3 dopamine and 5-HT 1A serotonin receptors but not for GABA or glutamate receptors. The inhibition of ligand binding to D 1 , D 2 , and D 3 dopamine and serotonin 5-HT 1A receptors was 100.6%, 98.41%, 70.63% and 79.3%, respectively (Table 1). cAMP assay for D 1

receptor activity
As shown in Fig 2A, the D 1 receptor agonist SKF 38393 but not l-THBr induced a dose-dependent increase in cAMP production in CHO cells stably expressing the D 1 receptor, with an EC 50 of 49.1 nM. In contrast, the D 1 receptor antagonist SCH23390 inhibited the production of cAMP induced by SKF 38393 (10 μM) in a dose-dependent manner with an IC 50 of 1.42 nM. l-THBr also inhibited cAMP production with an IC 50 of 361 nM (Fig 2B), indicating that l-THBr is a D 1 receptor antagonist. The maximum inhibition by l-THBr is 98.75% ±3.49. Significant binding affinity of l-THBr was defined as greater than 50% inhibition of ligand binding. The concentration of l-THBr and ligands was 10 μmol/L.

The effects of l-THBr in the light/dark box test
One-way ANOVA revealed significant differences among treatment groups (F (4, 43) = 6.068, P<0.001). The administration of DZP (2 mg/kg) or l-THBr (1 or 5 mg/kg) increased the number of transitions between the light/dark sides (P < 0.05 compared with the vehicle group). The results are shown in Fig 6. The effects of l-THBr in the elevated plus maze

Discussion
In the present study, we evaluated the anxiolytic-like effects of l-THBr in behavioral models of anxiety. We found that intraperitoneal administration of l-THBr produced anxiolytic-like effects in the elevated plus maze and light-dark box tests. In addition, l-THBr had a high affinity for D 1 , D 2 -like dopamine and serotonin 5-HT 1A receptors and exhibited D 1 , D 2 antagonist and 5-HT 1A agonist properties.
The anxiolytic mechanism of diazepam occurs mainly through benzodiazepine receptors, which are present in the GABA receptor pentameric complex. Thus, diazepam induces sedative effects by increasing the opening frequency of the associated chloride ion channel and hyperpolarizing the membrane [27]. In the present study, diazepam showed a significant and stable anxiolytic-like effect in the male ICR mice, consistent with some previous studies [28]. The in vitro receptor competitive test results demonstrated that l-THBr does not interact with inhibitory GABAergic receptors at benzodiazepine (BDZ) sites but mainly binds to dopamine and serotonin receptors. Thus, l-THBr works well at relieving anxiety without causing sedative effects.
Anxiety disorders are associated with the dysfunction of a number of neurotransmitters and their receptors, including dopamine and serotonin [29][30][31][32][33]. An increase in dopaminergic transmission has been demonstrated to aggravate anxiety [34], and the D 1 receptor antagonist SCH23390 exhibits clear anxiolytic-like effects [33,35,36]. However, D 2 receptor ligands can produce either anxiogenic [33,37] or anxiolytic-like effects in animal models [28,38,39]. D 1 dopamine receptors are mainly found at postsynaptic sites, whereas D 2 dopamine receptors are localized both presynaptically (where they act as autoreceptors) and postsynaptically. Therefore, D 2 antagonists may block presynaptic D 2 dopamine autoreceptors and increase the release of dopamine, which in turn modulate anxiety-like behaviors by acting on postsynaptic D 2 dopamine receptors [40][41]. Whether D 2 antagonists exert effects through presynaptic D 2 receptor or postsynaptic D 2 receptors may largely depend on the test doses used [39][40][41][42]. However, in our studies, the anxiolytic-like effect of l-THBr in the elevated plus maze test was blocked by the 5-HT 1A antagonist WAY100635. Thus, our results suggest that the anxiolytic-like effects of l-THBr are probably mediated through a 5-HT 1A receptor mechanism.
Previous studies indicate that injection of 5-HT into the brain stem produces anxiety [43]. Moreover, the anxiolytic activities of 5-HT 1A full or partial agonists are thought to be the result of decreased 5-HT outflow and a reduction of serotonergic neuron activity via the activation of 5-HT 1A autoreceptors at presynaptic sites [44].
In addition, the interaction of D 2 receptors and 5-HT 1A receptors plays an important role in mental disorders [45]. For example, the 5-HT 1A agonist buspirone at low doses of 1.25-5.0 mg/kg (which are relevant doses for the anxiolytic effects of buspirone) blocks presynaptic D 2 autoreceptors [46,47]. In addition, aripiprazole or SSR181507 (a combined D 2 antagonist and a 5-HT 1A partial agonist, respectively) improve depression and anxiety symptoms in patients with schizophrenia [48,49]. Based on these findings, it has been proposed that a combination of D 2 antagonistic and 5-HT 1A agonistic properties would offer additional advantages in treating some mental disorders, such as anxiety, depression (for fast onset anti-depressants) and schizophrenia [50,51].
In conclusion, l-THBr exhibits anxiolytic activity in two animal models of anxiety. Activation of 5-HT 1A autoreceptors and a decrease in serotonergic activity most likely contributes to the anxiolytic activity of l-THBr in these tests. The ability of l-THBr to exert effective anxiolytic activity without sedative effects suggests a potential use for l-THBr as a superior treatment for anxiety.

Author Contributions
Conceptualization: ZY HY BY JZ.