text:
Clinical and molecular characterization of 5α-reductase type 2 deficiency due to mutations (p.Q6X, p.R246Q) in SRD5A2 gene

. Early diagnosis and optimal management for steroid 5α-reductase type 2 deficiency (5α-RD2) patients are major challenges for clinicians and mutation analysis for the 5α-reductase type 2 ( SRD5A2 ) gene is the golden standard for the diagnosis of the disease. In silico analysis of this enzyme has not been reported due to the lack of appropriate model. Moreover, the histological and pathological changes of the gonads are largely unknown. In the present study, a 5α-RD2 patient born with abnormal external genitalia was studied and mutation analysis for SRD5A2 gene was conducted. Moreover, we constructed the homology modeling of 5α-reductase using SWISS-MODEL, followed by the molecular docking study. Furthermore, immunohistochemical staining of Ki67 for the testes tissue was conducted to investigate the potential pathological characteristics. The patient had male (46, XY) chromosomes but presented female characteristics, and the mutation analysis identified a heterozygotes mutation (p.Q6X, p.R246Q) in SRD5A2 gene. In silico analysis elucidated the potential effect of the mutation on enzyme activity. Immunohistochemical staining for the excised testes showed that 30%– 50% of the germ cells were Ki67 positive, which indicated the early neoplastic potential. In conclusion, we analyzed the genotype-phenotype correlations of 5α-RD2 caused by a heterozygotes mutation (p.Q6X, p.R246Q). Importantly, we conducted the homology modeling and molecular docking for the first time, which provided a homology model for further investigations. Immunohistochemical results suggested gonadectomy or testis descent should be performed early for 5α-RD2 patient, as delayed treatment would have maintained the testes in a tumorigenic condition.

ously, it is most important to diagnose DSDs correctly as soon as possible which will allow the patient to live as normal a life as possible. However, confirming a cause for DSD and devising a management plan is one of the most challenging clinical conditions for the clinicians.
One of the more unusual DSDs is 5α-reductase type 2 deficiency (5α-RD2). In this condition, although the affected individuals have male (46, XY) chromosomes, they usually present with ambiguous external genitalia at birth and are assigned female gender and raised up as girls [1,2]. As is shown in Fig. 1, it is caused by a deficiency of 5α-reductase isoenzymes, an enzyme that convert testosterone (T) into more biologically active dihydrotestosterone (DHT).
5α-RD2 is clinically and genetically heterogeneous. The clinical spectrum ranges from a total female appearance to a nearly complete male phenotype with mild symptoms of under-masculinization. Detailed genotypephenotype analysis may provide information necessary to improve diagnosis and treatment of 5α-RD2. 5α-RD2 is related to mutations in the 5α-reductase type 2 (SRD5A2, NM_000348.3) gene, which is located in chromosome 2p23 and contains five exons. So far, more than 100 different mutations have been reported in the SRD5A2 gene. However, it is difficult to investigate how the structure changes affect the enzyme activity as no homological model of the enzyme is available. Moreover, due to the low morbidity and different awareness of the disease, the histological and pathological changes of the genital system of 5α-RD2 patient are largely unknown. And it remains to be discussed which sex is suitable for a certain patient as most of the patients will be faced with a choice to be a male or female.
In the present study, we identified a DSD patient and made a detailed analysis of his clinical features, laboratory data, and management. Mutation analysis of the SRD5A2 gene was performed to make a molecular diagnosis. For the first time, homology modeling and molecular docking of 5α-reductase were carried out to investigate the effect of structural changes on the enzyme activity. Moreover, immunohistochemical staining of the testes tissue was conducted to investigate the potential histological and pathological characteristics of the genital system of 5α-RD2 patient. These results provide information necessary to improve early diagnosis and treatment of DSD for clinicians and better understand the genotype-phenotype correlations for 5α-RD2.

Patient and Methods
The study was approved by the Institutional Review Board of Shandong Provincial Hospital Affiliated to Shandong University (Jinan, China). All methods were carried out in accordance with the approved ethical guidelines and written informed consent was obtained from the patient and his parents.

Patient
A patient aged 15 years old was referred to our hospital (Shandong Provincial Hospital Affiliated to Shandong University, China) for the diagnosis and treatment of ambiguous genitalia. The patient was born with ambiguous genitalia and has been reared as a girl. She presented typical symptoms such as clitoris-like phallus, perineoscrotal hypospadias, cryptorchidism and undeveloped breast. There was no history of consanguineous marriage for three generations in the family.
Surgical treatments were carried out after the diagnosis confirmed, including bilateral testectomy, clitoridectomy and labiaplasty. The surgery was successful and the patient discharged from the hospital three days after the surgery. The patient was followed-up for one year, and the social gender, appearance, living conditions were recorded in detail.

Serum hormone studies
In order to facilitate the diagnosis of the patient, human chorionic gonadotropin (hCG, Livzon, China) stimulation test was carried out. The patient received daily intra-muscular injections of 1,500 IU of hCG for 3 days. The basal blood samples were taken on day 1 prior to administration of the first hCG dose, and the post stimulation samples were taken on day 4 for the measurement of T and DHT. Serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), T and DHT levels were measured by chemiluminescence immunoassay (Roche, Switzerland).

DNA extraction and amplification
Genomic DNA of the patient was extracted from peripheral leucocytes using genomic DNA kit (TIANGEN BIOTECH, Beijing, China, DP304-03). All five exons of the SRD5A2 gene were amplified by polymerase chain reaction (PCR) in a 25 μL system, which includes 2.0 μL dNTP, 2.5 μL 10 × PCR buffer, 0.15 μL Taq Hot Start (Takara Bio, Ohtsu, Japan), 2.0 μL genomic DNA and 0.5 μL 10 μM forward and reverse primers ( Table 1). The initial denaturation was 95°C for 3 min, subsequently followed by 35 cycles with denaturation at 94°C for 30 s, annealing at 70°C/55°C/52°C for 45 s and elongation at 72°C for 60 s. The amplifications were performed in an Eppendorf Mastercycler 5333 PCR amplification (Eppendorf Scientific, Germany).

Mutation analysis
PCR products were purified and sequenced in both the sense and the antisense directions on an ABI 3700 DNA sequencer (Applied Biosystems, PerkinElmer, Foster City, CA, USA). Sequences were compared with the reference sequence (NM_000348) by using CHROMAS software (v.2.01; Technelysium Pty Ltd, Tewantin, Qld, Australia). The mutation was named using the recommendations of the Nomenclature Working Group, in which the cDNA and protein sequence positions were designed by the prefixes c. and p. respectively.

Homology modeling and molecular docking
The NCBI protein database (http://www.ncbi.nlm.nih. gov/protein/) was used to search the amino acid sequence of the human 3-oxo-5-alpha-steroid 4-dehydrogenase 2 (5α-reductase). The employed protein sequence was NP_000339.2 reported by Hata. We applied methylomicrobium alcaliphilum delta(14)-sterol reductase (PDB ID: 4QUV) as the template, and the homology of amino acid sequence was aligned. Homology modeling of 5αreductase was carried out using SWISS-MODEL, a fully automated protein structure homology-modelling server. Molecular docking study was performed to investigate the binding mode between the NADPH, testosterone and the human 5α-reductase using Autodock vina 1.1.2. The 2D structures of the NADPH and testosterone were drawn by ChemBioDraw Ultra 14.0 and converted to 3D structure using ChemBio3D Ultra 14.0 software. The AutoDockTools 1.5.6 package was employed to generate the docking input files. The search grid of the 5αreductase for NADPH was identified as center_x:

Structural evaluation
The effect of the mutation Arg246Gln on the structure of the human 5α-reductase was evaluated by studying the homology modeling structure using PyMoL 1.7.6.

Immunohistochemistry (IHC)
Testicular morphology was assessed on haematoxylineosin (HE) staining, followed by immunohistochemical analysis for the expression and localization of Ki67. IHC was performed using paraffin-embedded tissue sections. The sections were dewaxed and hydrated, followed by antigen retrieval (in 0.01 mol/L citrate buffer solution, pH 6.0, heated to boiling for 2-3 min in a stainless steel pressure cooker). Endogenous peroxidase was blocked using a 3% hydrogen peroxide solution. The section was incubated with the blocking goat serum for 15 min, then immunostained with rabbit antibody against Ki67 (dilution 1:100; Abcam) at 4°C overnight. Secondary staining was performed with HRP-conjugated anti-rabbit IgG using a MaxVision Kit and a 3,5-diaminobenzidine (DAB) peroxidase substrate kit (Maixin Co, Fuzhou, China). The sections were then counterstained with hematoxylin, dehydrated, cleared, and mounted.

Clinical features and hormone studies
The patient came from a unconsanguineous family and was referred to our department due to genital dysplasia for 15 years. She was the only child of her family and her parents both presented normal phenotype. She had a female face and presented typical symptoms such as clitoris-like phallus, perineoscrotal hypospadias, cryptorchidism and undeveloped breast ( Fig. 2A-D). No other positive physical signs were shown. Pelvic ultrasonography presented no signs of uterus and accessories, while bilateral testes were shown within the prominence of the perineum. She has been raised as a girl since born.
Results showed that after hCG stimulation, the serum T level was almost doubled compared to the previous serum T level, while the serum DHT level only increased a little. The T/DHT ratio increased to 1.5 times compared with the basal ratio. Other detailed hormone levels of the patient were listed in Table 2. Combined with the aforementioned results, the primary diagnosis of the patient was 5α-reductase type 2 deficiency.

Management and follow-up
After the diagnosis of 5α-RD2 was confirmed by SRD5A2 gene mutation analysis, the patient received bilateral testectomy, clitoridectomy and labiaplasty ( Fig.  2E-F).
The patient was followed-up 1 month, 3 months, 6 month, 12 months after discharged from the hospital, respectively. Now the social gender of the patient is female, and the patient manifests slim shape and little body hair. She lives a normal female life by taking conjugated estrogens (0.3 mg/day, dosage adjusted according to the development of breast every month). She plans to undergo vaginoplasty in the near future.

Mutational analysis of SRD5A2 gene
In order to specify the diagnosis, the sequence analysis of the SRD5A2 gene was performed. The analysis revealed a heterozygotes mutation (Fig. 3A). A nonsense mutation (c.16 C > T, p.Q6X) was found in exon 1, and a missense mutation (c.737 G > A, p.R246Q) was found in exon 5. The former mutation derived from the mother of the patient, while the latter mutation came from her father. This result confirmed the primary diagnosis. As is shown in Fig. 3B, the missense mutation c.737 G > A affected the strictly conserved domains among vertebrate orthologs, while the nonsense mutation c.16 C > T leaded to a truncated protein.

In silico analysis of 5α-reductase
Till now, no research have reported how the structure changes affect the enzyme activity as no homological model of the enzyme is available. To further investigate the potential influence of the mutation on the functions of 5α-reductase, here we conducted homology modeling and molecular docking for the first time. Homology modeling of 5α-reductase was carried out using SWISS-MODEL. It was shown that human 5α-reductase features 6 α-helices (Fig. 4A). The structural impact of the Arg246Gln mutation was further investigated by study-ing the human 5α-reductase structure using PyMoL 1.7.6. Results showed that a change to glutamine at position 246 results in loss of electrostatic interactions with the NADPH (Fig. 4B-C).

Immunohistochemical studies
Gonadal tumors often occur in abnormal sexual development disorders and Ki67 is considered as a marker for the proliferative potential of tumor cells [3,4]. Therefore, we explored the expression of Ki67 in the testicular sections by immunohistochemistry to investigate the malignant potential of the testes of this patient. As shown in Fig. 5, germ cells with hyperchromatic nucleus could be seen in the tubules and germ cell maturation was occasionally observed. Moderate Ki67 staining cells  Clinical and molecular study of 5α-RD2 accounts for more than 50% in the left testis tissues and 30% in the right testis of the patient. These results indicated the early neoplastic potential for the testes of this patient. No interstitial fibrosis was observed between the oval seminiferous outlines.

Discussion
In the present study, a 5α-RD2 patient born with abnormal external genitalia was recorded in detail. Mutational analysis of SRD5A2 gene was conducted and etiological mutations (p.Q6X and p.R246Q) were found. Moreover, we conducted the homology modeling and molecular docking for the first time, which provide a homology model for further investigations. Furthermore, the histological changes of the genital system was investigated, which may contribute to direct and improve the treatment of the disease.
The clinical manifestations of the patient here is typical, including clitoris-like phallus, perineoscrotal hypo-spadias, cryptorchidism and undeveloped breast [1,2,[5][6][7]. In order to identify the patient from diseases that with the similar clinical manifestations such as androgen insensitive syndrome (AIS) and Leydig cell hypoplasia, hCG stimulation test was performed. Results showed a significant elevation of serum T/DHT ratio, indicating that the testes of the patient were able to secret T and the conversion from T to DHT was suppressed. However, the post hCG stimulation T/DHT ratios varies according to age and the severity of the enzyme defects, and the false-negative results [8][9][10][11]. There are patients that diagnosed with 5α-RD2 by gene mutational analysis even they had normal T/DHT ratio after the hCG stimulation, which means the precise diagnosis was not accomplished for this patient [9,12].
Importantly, we conducted the homology modeling of 5α-reductase using SWISS-MODEL, followed by the molecular docking study was performed to investigate the binding mode between the NADPH, testosterone and the human 5α-reductase. The detailed mechanisms of how the mutations affect the enzyme activity of 5αreductase has never been investigated due to the lack of homology model. Our in silico results here showed that the residue Arg-246 is located in the loop region near the C-terminal, and forms electrostatic interaction with the NADPH. The substitution of the polar amino acid arginine for neutral glutamine in the protein will lead to a severe loss of enzyme function. As far as we know, this is the first report on the homology modeling and molecular docking of human 5α-reductase, which provides a homology model for further investigations.
Moreover, we should notice that gonadal tumors often occur in abnormal sexual development disorders. Due to the increased level of gonadotropin and maldescent of testes, 5α-RD2 patients who are assigned male gender or received surgery during their elder age may have high risk to develop gonadal tumors. Ki67 is a nuclear protein that expressed by cells in G1, S, G2, and M phases of the cell cycle. It is rapidly metabolized after M phase and is not synthesized in G0 phase. Therefore, the expression of Ki67 reflects the proliferative potential of tumor cells [3,4]. Here we performed the Ki67 immunohistochemical staining for the surgical specimens of the patient. Importantly, the immunohistochemical results showed that the Ki67 positive germ cells account for 30%-50% of all the germ cells in the testes tissues of the patients, significantly higher than that in the normal testis, which reveals a potential in developing gonadal tumors for this patient. It has been reported that the risk of gonadal tumors in 5α-RD2 patients is low, this may due to the limited data and cases [13]. Theoretically, the malignant potential of gonadal tissues in 5α-RD2 patients should be further investigated in detail. As the testes of these patients are usually hypofunctional, which subsequently results to an increased gonadotropin level. Gonadal tumors tend to occur in degenerative testes and augmented gonadotropin stimulation has been demonstrated to be important in gonadal tumors' development [14,15]. In addition, as testicular maldescent is common in 5α-RD2 patients, the increased temperature within the intra-abdominal environment also increased the susceptibility to the development of gonadal tumors [14,16]. Accordingly, a giant A: Total view of the mutation. B: 5α-reductase wt: The yellow dotted lines show the electrostatic interactions between the side chain of Arg-246 (green) and NADPH (salmon). C: 5α-reductase Arg246Gln: In the mutant protein, the electrostatic interaction between Gln-246 (green) and NADPH (salmon) is lost. The figures were generated from the human 5α-reductase using PyMoL 1.7.6 to visualize the structural effect of mutation.
Clinical and molecular study of 5α-RD2 seminoma in an adult Japanese phenotypic female who was homozygous for the p.Q6X substitution was reported, demonstrating that the potential of developing gonadal tumor in 5α-RD2 patients should be paid more attention [17].
It has been reported that most of the patients with 5α-RD2 are typically first assigned a female gender, and due to the significant masculinization, psychosexual orientation and cultural biases, a number of these patients may switch to male gender during puberty [18,19]. Our aforementioned immunohistochemical results suggested that gonadectomy should be performed when the female gender is chosen, and early testis descent is necessary when the male gender is assigned, as delayed treatment would have maintained the testes in a tumorigenic condition.
Generally, genetic analysis is essential for the diagnosis and treatment of patients with gonadal abnormalities. Thanks to the genetic analysis, a number of etiologic mutational genes have been identified, such as SF1, StAR, HSD3B2 and CYP17 [20,21]. As for SRD5A2, more than 100 different mutations scattered throughout the five exons have been reported in different ethnic groups so far. Over 90% of all the mutations are missense mutations, nonsense mutation, deletions and insertions. It has been reported that more than 60% of patients were homozygotes and others were heterozygotes [22][23][24][25][26]. Here we also performed mutational analysis of SRD5A2 gene, the results revealed two different mutations (p.Q6X and p.R246Q). Both of these mutations have been identified previously. The p.Q6X mutation was a common nonsense mutation that has been reported mainly in Asiatic patients from Thailand, Korean and Chinese origins [17,[27][28][29]. This mutation results in the formation of completely truncated protein missing the binding sites to T and its cofactor [17,27,[29][30][31][32]. The p.R246Q is a missense mutation that impairs 5αreductase activity due to a reduced NADPH binding Immunohistochemical staining of the testes using the anti-Ki67 antibody. [33]. It has been reported that the p.R246Q mutation is common among patients with SRD5A2 gene defect from the Northern states of India. Ambiguous genitalia is the common clinical manifestation, including enlarged clitoris or clitoris-like underdeveloped glans, perineal hypospadias, microphallus and labial fusion. Spontaneous virilization usually emerges at puberty [27,34]. The detailed data of related patients were summarized in Table 3 [19,[27][28][29][35][36][37].
There are several issues need to be further investigated. We should pay more attention to the histological and pathological changes of the genital system of 5α-RD2 patients. A long time follow-up is needed to present the outcomes of 5α-RD2 patients that assigned different genders.
In conclusion, we analyzed the genotype-phenotype correlations of 5α-RD2 caused by a heterozygotes mutation (p.Q6X, p.R246Q) in detail. Our research emphasized the importance of mutation analysis of SRD5A2 gene in diagnosing the disease. Importantly, we conducted the homology modeling and molecular docking for the first time, which provided a homology model for further in silico investigations on 5α-reductase. Moreover, immunohistochemical results suggested gonadectomy or testis descent should be performed early for 5α-RD2 patient, as delayed treatment would have maintained the testes in a tumorigenic condition. All these results emphasized the importance of early diagnosis Clinical and molecular study of 5α-RD2 and surgical treatment, which may benefit the final prognosis of the 5α-RD2 patients.

Funding
This work was supported by grants from the National Natural Science Foundation (81370891 and 81670720), special funds for Taishan Scholar Project (tsqn20161071).

Disclosure
There are no conflicts of interest to disclose.


extfieldsofstudy: Chemistry Medicine

text:
Modulation of Neuronal Activity on Intercalated Neurons of Amygdala Might Underlie Anxiolytic Activity of a Standardized Extract of Centella asiatica ECa233

GABAergic intercalated neurons of amygdala (ITCs) have recently been shown to be important in the suppression of fear-like behavior. Effects of ECa233 (a standardized extract of Centella asiatica), previously demonstrated anxiolytic activity, were then investigated on ITCs. Cluster of GABAergic neurons expressing fluorescence of GFP was identified in GAD67-GFP knock-in mice. We found that neurons of medial paracapsular ITC were GABAergic neurons exhibiting certain intrinsic electrophysiological properties similar to those demonstrated by ITC neurons at the same location in C57BL/6J mice. Therefore, we conducted experiments in both C57BL/6J mice and GAD67-GFP knock-in mice. Excitatory postsynaptic currents (EPSCs) were evoked by stimulation of the external capsule during the whole cell patch-clamp recordings from ITC neurons in brain slices. ECa233 was found to increase the EPSC peak amplitude in the ITC neurons by about 120%. The EPSCs in ITC neurons were completely abolished by the application of an AMPA receptor antagonist. Morphological assessment of the ITC neurons with biocytin demonstrated that most axons of the recorded neurons innervated the central nucleus of the amygdala (CeA). Therefore, it is highly likely that anxiolytic activity of ECa233 was mediated by increasing activation, via AMPA receptors, of excitatory synaptic input to the GABAergic ITC leading to depression of CeA neurons.

Introduction
Emotional symptoms especially fear/anxiety-like behavior are considered to be regulated through the amygdala region. The stimuli from the environment reach the basolateral amygdala (BLA) via thalamic and cortical afferents and transmit to the central nucleus of amygdala (CeA) where a proper anxiogenic response occurs [1]. Between BLA and CeA, the relay local processing happens at the intercalated cells (ITCs) level. The ITCs of the amygdala consist of densely packed GABAergic neurons surrounding BLA providing inhibitory control of neurons in BLA and CeA [2]. This group of neurons receives information from both intra-and extra-amygdaloid nuclei. The external capsule is the major connecting fiber to the ITCs. However, the exact fiber in external capsule that inputs to the ITCs is not clearly identified [3,4]. Nevertheless, the projections from the medial prefrontal cortex to ITCs are thought to be a critical component of the forebrain circuitry for fear extinction [3]. Furthermore, ablation of ITCs has led to extinction retrieval deficits indicating the requirement of ITCs for the expression of fear extinction [5]. Thus, previous data suggest that ITCs play a critical role in fear extinction; a very important translational process since extinction-based exposure therapies is the mainstay for the treatment of anxiety disorder [6]. Therefore, it is our interest to explore how ITCs would respond to ECa233, a standardized extract of Centella asiatica previously shown to possess anxiolytic activity by our group [7].

Evidence-Based Complementary and Alternative Medicine
Centella asiatica (CA), one of psychoactive medicinal plants, has been used in Ayurvedic medicine for centuries to relieve the symptom of anxiety and to contribute a deep state of relaxation and/or mental calmness throughout meditation practices. A recent clinical study showed the beneficial effects of CA on generalized anxiety disorder [8].
As to the mechanism of its action in brain function, CA is considered to enhance the GABAergic neurotransmission at the presynaptic level by facilitating the glutamic acid decarboxylase activity [9]. However, the possible function of CA on the glutamatergic synaptic transmission to GABAergic neurons remained unclear. A standardized extract of Centella asiatica or ECa233 was prepared to avoid the large variation in composition of herbal extracts and to ensure consistency in physical and chemical constituents. ECa233 contained triterpenoid glycosides not less than 80% and the ratio between madecassoside and asiaticoside should be 1.5 ± 0.5. Previous studies demonstrated that ECa233 exerted the neuroprotective effects on learning and memory deficit induced by 2-vessel occlusion (2VO) in mice at the doses of 10 and 30 mg/kg [10]. It protected the neuronal cell death in hippocampus. Moreover, the anxiolytic activity of ECa233 on stress-induced anxiety in chronic immobilization models was observed [7]. Interestingly, the effect of ECa233 on ERK1/2 signaling pathways was reported to promote the neurite outgrowth of neuronal cells [11].

Animals.
The protocol of animal housing and treatment used in this study was approved by the Ethics Committee of the Faculty of Pharmaceutical Sciences, Chulalongkorn University, Thailand, and the Institutional Animal Care and Use Committee of the National Institutes of Natural Sciences, Japan. The GAD67-GFP knock-in and wild type C57BL/6J mice aged between 14 and 21 days were used for slice preparation. The procedures for the generation and genotyping of the GAD67-GFP knock-in mice were described by Kaneko et al., 2008 [12]. Mice homozygous for the GAD67-GFP allele were mated with C57BL/6J wild type mice to obtain the heterozygous mice. Wild type C57BL/6J mice were obtained from the National Laboratory Animal Center, Mahidol University.
The procedures of whole cell patch-clamp recordings are described previously [13]. Whole cell patch-clamp recordings were conducted on the neurons in ITC under visual control of patch pipettes using a patch-clamp amplifier (EPC-7; List, Darmstadt, Germany). GFP-positive neurons were selected using epifluorescence microscopy and then visualized with Nomarski optics with ×63 water-immersion objective lens. Patch pipettes were pulled from borosilicate glass capillaries (GC150T-10, 1.5 mm OD, Harvard Apparatus, Kent, UK) using the horizontal electrode puller (P-97; Sutter Instrument, CA, USA). The pipettes were filled with a gluconatebased intracellular solution containing (in mM): 140 Kgluconate, 20 KCl, 0.2 EGTA, 2 MgCl 2 , 2 Na 2 ATP, 0.5 Na 3 GTP, 10 HEPES, and 0.1 spermine (pH 7.3) for the study of firing pattern, passive, and active properties of neurons. The Cs-based intracellular solution was used to study the AMPA and NMDA receptor-mediated excitatory postsynaptic currents, effect of ECa233 on eEPSCs consisting of (in mM): 120 Cs-gluconate, 10 CsCl, 2 MgCl 2 , 4 Na 2 ATP, 10 EGTA, 10 HEPES, and 0.1 spermine (pH 7.3). To stain the recorded neurons, biocytin (4 mg/ml; Sigma, St. Louis, MO) was dissolved in the intracellular solution. As the liquid junction potential between the standard Ringer's solution and the gluconate-based intrapipette solution was estimated at −10 mV, the actual membrane potential was corrected by this value. The osmolarity of the intrapipette solution was 280-290 mOsm/L. The resistance of the electrodes is 5-8 MΩ in the bath solutions. All recordings were performed at 32-34 ∘ C. Data were acquired using the pClamp system (pClamp 8.1; Molecular Devices, CA, USA).

Comparative Electrophysiological Properties of ITCs between GAD67-GFP Knock-In and Wild Type C57BL/6J
Mice. Initially electrophysiological recordings were made on GABAergic neurons of ITC, easily identified by GFP fluorescence, in GAD67-GFP knock-in mice. Depolarizing current pulses were applied with 400 ms duration and step amplitudes up to 200 pA (40 pA in each step). The resting membrane potential (RMP), input resistant (Rin), threshold, and action potential amplitude were recorded. The same procedure was applied on neurons at the same location in wild type C57BL/6J mice and similar electrophysiological characteristics were obtained indicating GABAergic properties of the recorded neuron which were further used for analysis of ECa233 activity on ITC of amygdala.

Effect of ECa233 on EPSCs of ITCs.
Electrical stimulation of external capsule was applied as a cathodal square wave pulse of 200 s duration with an intensity of up to 150 A using a concentric bipolar electrode (Clark Electromedical Instruments, Pangbourne, UK) to activate the input fibers to ITCs (Figures 1 and 2). ECa233 was bath applied to examine the EPSCs on ITCs. Data was analyzed with Clampfit Evidence-Based Complementary and Alternative Medicine 3  (Molecular Devices, CA, USA). Statistical significance was examined with a two-tailed Student's paired -test and the difference was considered significant if < 0.05. To visualize the recorded neurons, 4 mg/ml biocytin was dissolved in the intrapipette solution and diffused into the neuron during the patch-clamp recordings. In order to distribute biocytin throughout the recorded neuron (estimated to take 30 min), slices were fixed >30 min. The patch pipettes were carefully detached from the cells after recording. The slices were fixed with 4% paraformaldehyde in 0.12 M phosphate buffer, pH 7.4, for 2-3 days at 4 ∘ C, rinsed in 0.05 M PBS, pH 7.4, and incubated in methanol containing 0.6% H 2 O 2 for 30 min. After washes with PBS, the slices were incubated in avidin-biotin peroxidase complex solution (1%) (Vector Laboratories, Burlingame, CA) containing 0.3% Triton X-100 for 3 h. After washes with PBS and 0.05 M Tris-buffered saline (TBS), pH 7.6, they were incubated in a TBS solution containing 0.01% diaminobenzidinetetrahydrochloride (DAB), 1% nickel ammonium sulfate, and 0.0003% H 2 O 2 for 30 min. All procedures for visualization of biocytin were performed at room temperature. The slices were mounted on gelatincoated slides, counterstained with cresyl violet, dehydrated, and then coverslipped. The morphological properties of stained cells were drawn using a camera lucida attached to a light microscope (Nikon, Japan).

Statistical Analyses.
Results are represented as mean ± SEM from eight to ten animals per group. Statistical differences of the mean were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett's post hoc test for a significance level when the " " value was less than 0.05. Simple comparison between two groups was performed using Student's -test. All statistical analyses were performed using PASW Statistics 18 (SPSS Inc, Chicago, USA).

The Morphological and Electrophysiological Properties of ITCs in GAD67-GFP Knock-In and Wild Type C57BL/6J Mice.
Firstly, we use GAD67-GFP knock-in mice to identify the GABAergic neurons in the ITCs. We found that ITC was a well-established structure with clear boundaries and almost all were GABAergic neurons. Comparative structure between GAD67-GFP knock-in and wild type C57BL/6J mice was shown in Figure 2. The boundary of this area was also visible in wild type C57BL/6J mice. Electrical properties of ITC neurons in GAD67-GFP knock-in and wild type C57BL/6J mice were investigated. The firing pattern and morphological properties of neuron in ITC between GAD67-GFP knock-in and wild type C57BL/6J mice showed no differences (Figure 3). Three types of firing pattern were demonstrated: regular spiking, late spiking, and fast spiking. The morphological properties were typically bipolar with the I-V plots of input resistance (Figures 3(c1) and 3(f1)). The passive and active membrane properties of neurons in both mice showed no difference ( Table 1). The value of RMP, Rin, threshold, and action potential amplitude was 0.195, 0.832, 0.565, and 0.586, respectively, by Student's pair -test.

AMPA/NMDA Receptors Mediated eEPSCs Recorded in the ITC.
In this study, the stimulating electrode was placed on the external capsule and the recording electrode was patched on neurons in the ITCs cluster (Figures 1 and 2). In order to examine the voltage dependence of the evoked      EPSCs (eEPSCs), recording was performed in the presence of bicuculline (10 M) to block GABA A receptor activity and the Cs-based intracellular patch solution was used to reduce outward potassium current and block GABA B receptors. The ionotropic glutamate receptor-mediated component at these synapses had a linear current-voltage ( -) relationship and reversal potential of +1.2 mV (Figures 4(a) and 4(b)). NMDA-EPSCs were isolated by blocking AMPA/kainate receptor with CNQX (50 M) in the presence of bicuculline. The NMDA receptor-mediated component at these synapses had a nonlinear -relationship, with a region of negative slope between −40 and +10 mV and reversal potential of 7.8 mV (Figures 4(a) and 4(c)). Additionally, the application of 10 M APV (a selective NMDA receptor antagonist) in the presence of bicuculline and CNQX totally abolished the response (Figures 4(a) and 4(d)).

ECa233 Increased the Amplitude of eEPSCs in ITC.
To study the effect of ECa233 on the eEPSCs, we continuously recorded the eEPSCs in ITC neurons every 2 min for 1 h. We found that the eEPSCs gradually increased 10 min after initiation of bath application of ECa233 (Figure 5

Concentration-Response Curve of ECa233.
To characterize the excitatory effects of ECa233 on ITC neurons, the concentration-response curve of ECa233 was obtained by testing the effects of different concentrations of ECa233 (Figure 6). ECa233 (0, 0.03, 0.1, 0.3, 1.0, and 3.0 g/m) generated a concentration-dependent enhancement of EPSCs in ITC neurons. The estimated maximal increase of eEPSCs was 120%, and the estimated value of EC 50 was 0.25 g/ml. Effects of ECa233 were completely abolished after being washed by PBS for 30 min. Example tracing of eEPSCs before and after a bath application of ECa233 (1 g/ml) for 15 min was shown in the inset of Figure 6. All of the solutions contained 10 M bicuculline.

Effect of ECa233 in the Presence of Blockers of Fast Synaptic
Transmission. We examined the ECa233-induced increase of eEPSC amplitudes in ITC neurons in the presence of various   (Figure 7).

Discussion
In the present study, bath application of ECa233 significantly increased evoked EPSCs of ITCs up to 120%. Further experiments conducted in the presence of a series of antagonist(s) suggested that modulation of AMPA receptors was responsible for the increased evoked EPSPs observed and ultimately leading to anxiolytic activity in animal models. To our knowledge, this is the first report to demonstrate a modulation of ITCs of amygdala as possible mechanism underlying anxiolytic activity of a natural product, the standardized extract of Centella asiatica ECa233 in animal models. Furthermore, our findings could possibly suggest the prominent role played by ITCs in fear extinction as a potential pharmacological treatment of anxiety as well.
Due to the lack of supply of GAD67-GFP knock-in mice in Thailand and our findings that the morphological and electrical properties including active and passive membrane properties of specific cluster of neurons in GAD67-GFP knock-in and C57BL/6J mice were not different [14,15], we conducted experiments using C57BL/6J mice to study the effect of ECa233, a natural extract of Centella asiatica, on ITCs neurons.
Stimulation of ITCs was made by a concentric bipolar electrode placing on external capsule encompassing a number of fibers from various nuclei including mPFC-ITCs pathway responsible for anxiolytic-like behavior seen in elevated plus maze and open field test [7]. Recently, there were numerous studies of fear extinction-based therapies for the treatment of anxiety disorder focusing on the mPFC-ITCs pathway. The density of mPFC fibers innervated the ITCs is a subject of controversy. Several laboratories indicated that the mPFC provides a direct innervation to ITCs [3,16]. However, Strobel and their coworker showed the di-synaptic of mPFC-ITCs via the BLA [4]. Therefore, at the moment, anxiolytic activity of ECa233 acting on ITCs may or may not involve mPFC-ITCs pathway. Synaptic inputs of ITC neurons coming from the external capsule are glutamatergic synapses similar to the result of main intercalated cells [14], suggesting that long term depression and potentiation can occur in the ITCs. It is well known that CeA promotes the anxiogenic-like behaviors. For example, the injection of muscimol was found to impair the avoidance of rats to the open arms of the elevated plus maze [17]. So, the strongest feed forward inhibition of ITCs to CeA should present the anxiolytic activity. ECa233 increased the eEPSCs in ITCs via the AMPA receptors as mentioned in this experiment. The finding that brain slices had to be perfused with ECa233 in 5-10 min before the effects were detected suggested the possibility that ECa233 might increase the phosphorylation of AMPA receptor. Previous study of our group demonstrated the upregulation of ERK1/2 and PI3K/Akt signaling pathways after ECa233 exposure [11]. Increased PI3K/AKT signaling was associated with increased expression of AMPA receptors in the surface membrane [18]. In addition, superfusion of ECa233 for 10 min in brain slice preparation increased the long-term potentiation and the BDNF protein expression in hippocampus (unpublished observation). Unfortunately, in the present study, no attempt is made to probe for a presynaptic or postsynaptic site of action for ECa233. This topic should be further investigated in the future.
In line with other studies, the collateral axon of ITCs in the present study mainly innervates CeA [2,15] suggesting that facilitation of ITCs activity by ECa233 could therefore lead to an increased GABAergic inhibition of CeA. GABA is known as major inhibitory neurotransmitter of the brain and dysfunction of GABAergic neurotransmission causes neurological disorder. Thus, activities of ECa233 on other neuronal circuitry using GABA are plausible and await proof of evidence.
Taken all together, it is highly likely that ECa233 modulated the AMPA receptor and consequently activated ITC neuron to release GABA neurotransmitter in CeA, which 8 Evidence-Based Complementary and Alternative Medicine then suppressed the CeA neuronal activity. Thus, it is possible that ECa233 acutely reduced the anxiety behavioral responses in animal models by increasing the GABA release in CeA via modulation of ITC neurons. Similar results were previously reported on neuregulin-1, the neurotrophic factor that could u-regulate AMPA receptor in GABAergic neurons and hence explained its benefit in the treatment of mental disorder [19].

Conclusions
The present study demonstrated the mechanism of anxiolytic activity of ECa233, a standardized extract of Centella asiatica, on ITCs of the amygdala. ECa233 facilitates the AMPA receptor-mediated excitatory synaptic inputs in ITCs. Since most of the ITC axons innervated CeA. We suggest that increasing the excitatory responses of ITCs by ECa233 would promote the GABA release in the CeA suppressing the output signals from the amygdala.

Disclosure
Tadashi Isa present address is as follows: Department of Neuroscience, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.

Conflicts of Interest
The authors declare that they have no conflicts of interest.