Journal of Research in Pharmacy

Summary

γ-amino bütirik asit aracılığıyla oluşan sinir iletisi, anksiyete, uyku bozukluğu, depresyon ve bipolar hastalık gibi durumların tedavisinde kullanılmaktadır. Bu çalışmanın amacı Wistar albino sıçanlara akut lamotrijin uygulamasının, beyin omurilik sıvısındaki GABA ve L-glutamik asit düzeylerine nasıl etki ettiğine ve olası etkilerin kolinerjik sistemle ilişkisine dair /nörokimyasal kanıt sağlamaktır. Konsantrik mikrodiyaliz problarının lateral ventriküllere yerleştirilmesinden bir gün sonra uyanık hayvan modelinde mikrodiyaliz deneyleri yapıldı. Sıçanlara fizyolojik tuzlu su veya 20 mg/kg lamotrijin uygulaması yapıldı. Kolinerjik katılımı göstermek için 0.5 mg/kg fizostigmin veya 2 mg/kg atropin sülfat ön uygulamaları lamotrijin enjeksiyonundan önce uygulandı. Toplanan diyalizatlarda GABA, L-glutamik asit ve lamotrijin düzeyleri yüksek performanslı sıvı kromatografisi ile analiz edildi. Fizyolojik tuzlu su, GABA veya L-glutamik asit düzeyinde bir etki oluşturmazken, lamotrijin anlamlı derecede GABA düzeyini artırdı (p<0,05). Fizostigmin veya atropin ön uygulamaları lamotrijinle indüklenen GABA atışı üzerine bir etki oluşturmadı. Bu sonuçlar lamotrijinin farmakolojik etkilerinde GABA'nın katılımının olduğunu ve aralarında lineer bir ilişki bulunduğunu göstermektedir. Santral kolinerjik sistem lamotrijin ile indüklenen bu etkiye katılmamaktadır.

Summary

Introduction

Lamotrigine as a comparatively novel antiepileptic agent is now being used widely as a drug of first choice in certain seizure types including Lennaux Gestaut syndrome, grand or petit mal seizures and myoclonus[1-8]. Lamotrigine has also been extensively preferred for bipolar disorders[9,10,11]. γ-amino butyric acid (GABA) is the major inhibitory neurotransmitter in the brain and low GABAergic activity has been implicated in the pathophysiology of bipolar disorder[12,13]. Previous studies indicated that GABAergic potentiation plays role in the effects of lamotrigine in addition to its anti-glutamergic effects[14,15].

Lamotrigine reduces glutamate and aspartate release through inhibiting Na⁺ channels and thus causing inhibition of exocytosis of these excitatory amino acids[16]. The role for inhibition of Ca²⁺ channels was also demonstrated[17-19].

Prior studies performed in rat entorhinal cortex using whole patch clamped cells demonstrated that lamotrigine produces reductions in glutamate and increases in GABA release without affecting Na+ or Ca²⁺ channels[20,21]. It was also reported that lamotrigine also suppresses GABAA-mediated neurotransmission in rat amygdala cells through affecting presynaptic Ca²⁺ influx[22]. Lamotrigine also decreases veratrineor electrically stimulated release of endogenous glutamate and [3H]-GABA, [3H]-5-hydroxytriptamine and [3H]-dopamine in rat cortical slices[17]. However, ex vivo studies documented that acute lamotrigine did not produce an effect on hippocampal tissue content of GABA or taurine but both were increased following chronic treatment with the drug[23].

In central nervous system, the interaction between cholinergic system and GABAergic transmission is long studied. Previous in vivo and in vitro studies also demonstrated that GABA and its analogues directly inhibit cortical acetylcholine release in the freely moving guinea pig and in electrically stimulated slices[24,25]. Local inhibitory effects of acetylcholine have been ascribed to the excitation of GABAergic inter neurons in the cerebral cortex[26] but some findings suggested that acetylcholine exerts direct inhibitory effects on GABA release at least in other brain areas[27,28] and in the periphery[29].

The aim of this study is to monitor the time-course changes of GABA and L-glutamic acid in rat cerebrospinal fluid produced by lamotrigine treatment by measuring the amino acids dialyzed through microdialysis probes implanted into lateral ventricles of conscious rats and secondly to show the possible modulatory effect of cholinergic system on the lamotrigineinduced amino acid release.

Introduction

Methods

1. Animals and laboratory
Wistar albino rats weighing 250-275 g of both sexes supplied from Marmara University, Experimental Research and Animal Laboratory were used. An approval of Marmara University Ethical Committee for Experimental Animals was taken before the experiments (16.12.2005 - 63.2005.mar). The animals were kept in a temperature-controlled room with 12-h light and dark cycle and fed with standard animal food and water ad libitum.

2. Drugs used in the study
All drugs were supplied from Sigma Chemical (USA) except lamotrigine (supplied kindly from GlaxoSmithKline, Turkey). Lamotrigine was dissolved physiological saline prior to injection.

3. Stereotaxic surgery and microdialysis
Concentric microdialysis probes were used as as described previously[30]. The rats were anesthetized with intraperitoneal ketamine (100 mg/kg) and chlorpromazine (1.0 mg/kg) mixture and placed in a stereotaxic frame (Stoelting, Model 51600, USA). The scalp skin was incised and the periosteum was separated from the cranium. Probe was implanted into the right lateral ventricle (lateral ventricle coordinates; 1.0 mm posterior to bregma, 1.5 mm lateral to midline and 3.8 mm ventral to the skull surface) according to the Paxinos and Watson rat brain atlas[31]. Supporting screws were also placed and the microdialysis probe was covered together with the screws with dental acrylic cement. The collection of intracerebral perfusion samples was performed 24 h following surgery.

The day after the placement of microdialysis probes, polyethylene tubings were attached to the inlet of the microdialysis probes to collect the samples in conscious rat model in a plexyglass cage (42X42X20 cm). Artificial cerebrospinal fluid was delivered continuously via 250 μl hamilton syringe which was connected to a microinfusion pump (KD Scientific, USA). The composition of artificial cerebrospinal fluid was 2.5 mM KCl, 125 mM NaCl, 1.26 mM CaCl₂2H₂O, 1.18 mM MgCl₂6H₂O and 0.2 mM NaH₂PO₄2H₂O and the pH was set to 7. The artificial cerebrospinal fluid was filtered through 0.4 μm nylon membrane filters.

Two basal samples were collected at 0.5 μl/minute flow rate every 40 min in a 0.25 ml ependorf tubes from Wistar rats after an equilibration period of 1 h. After collection of basal samples, intraperitoneal physiological saline injection was administered and five more consecutive samples were collected. The same protocol was repeated with lamotrigine (20 mg/kg), physostigmine (0.5 mg/kg) or atropine sulfate (2 mg/kg). The dialysates were divided into two equal ependorf tubes for different High Performance Liquid Chromatography analysis methods and kept at -80ºC.

Throughout the microdialysis procedure, the rats were observed and atypical behaviors were noted. The rats were anesthetized with ether and methylene blue was injected through the probe and decapitated. The brains were sliced with a blade to observe the dye in the ventricles for verification of probe placement. Only the proper experiments were used in data analysis.

4. Chromatographic system and High Performance Liquid Chromatography analysis of L-glutamic acid and GABA in the cerebrospinal fluid dialysates
Chromatographic system for analysis of L-glutamic acid and GABA (supplied from Sigma, USA) consists of a gradient pump (Agilent 1100, Germany) with four solvent bottles, degasser module, C18 reverse phase nucleosil column (15 cm and 3.9 cm length, 4.6 mm diameter and 5 μm pore size), autosampler unit, fluorescent detector with excitation and emission wave lengths set to 360 nm and 410 nm respectively and a computer. The composition of mobile phase and chromatographic procedures were performed as described previously[32].

5. Lamotrigine and amino acid High Performance Liquid Chromatography analysis
The isocratic High Performance Liquid Chromatography system for lamotrigine analysis consists of a 100 μl loop, rheodyne valve with a pump (Jasco PU 980, Tokyo, Japan), C18 reverse phase colon (15 cm length, 4.6 μm diameter and 5 mm pore size), UV detector (Jasco UV 975, Tokyo, Japan) wave length set to 214 nm and a computer. The chromatographic analysis was carried out with a software (Borwin Chromatograph, version 1.2, France). The mobile phase is a mixture of 0.1 M KH- ₂PO₄ (pH: 6.7), acetonitrile and methanol (7:2:1, v/v/v). The flow rate of the pump was set to 1.3 ml/min. Manual injections were given within a volume of 10 μl at room temperature. The retention time of lamotrigine was 4.5 min. Total duration of the chromatogram was 15 min.

6. Statistical analysis
All data are expressed as means ± s.e.m. The effect of saline or drug treatment on amino acids were tested using Kruskal-Wallis followed by Dunn's Multiple Comparison Test. The relationship between the lamotrigine and GABA concentrations in the CSF dialysates was determined by Pearson's test (alpha = 0.05). Two-tailed Student's t-test for unpaired data was used to determine the differences between the basal percent change of GABA after physostigmine or atropine sulfate injections. Statistical significance was accepted where p<0.05.

Methods

Results

1. The effect of lamotrigine injection on GABA and L-glutamic acid in cerebrospinal fluid dialysates
The basal levels of L-glutamic acid and GABA in the cerebrospinal fluid dialysates prior to physiological saline injection were found to be 2.35 ± 0.42 mM and 0.16 ± 0.01 mM, respectively. Physiological saline injection produced no significant difference either in L-glutamic acid or GABA levels (Figure 1A and 1B; p=0.565 and p=0.789). Lamotrigine injection did not affect L-glutamic acid levels (Figure 1C; p=0.922) but produced increases in GABA level yielding significant differences in [40- 80]-, [80-120] - and [120-160]-min sampling intervals when compared to [-40-0]-min (basal) sample (p<0.001, p<0.001 and p<0.01, respectively; Figure 1D).

Click Here to Zoom FIGURE 1: The effect of intraperitoneal physiological saline injection on L-glutamic acid (A) and GABA (B) levels in cerebrospinal fluid dialysates collected from the right lateral ventricle of Wistar rats (n=10). Lamotrigine (LTG) was administered at a dose of 20 mg/kg and its effects on L-glutamic acid and GABA are presented in C and D, respectively (n=10). *p<0.01 **p<0.001 (compared to [-40-0]-min (basal) sample)

2. The relationship between GABA and lamotrigine levels in the cerebrospinal fluid dialysates
Lamotrigine started to appear in [0-40] min samples following intraperitoneal injection, and reached a peak at [80-120]-min period and the levels started to decline in the samples collected afterward (Figure 2). When GABA and lamotrigine concentrations measured within the same sampling periods are plotted and a linear relationship between the drug and GABA was recognized (Figure 3; p=0.0122, alpha=0.05).

Click Here to Zoom FIGURE 2: The time course change in lamotrigine (LTG) concentrations in cerebrospinal fluid dialysates following intraperitoneal injection. *p<0.01 **p<0.001 (compared to [-40-0] min (basal) sample)

Click Here to Zoom FIGURE 3: The relationship between GABA and lamotrigine (LTG) levels in the cerebrospinal fluid dialysates. Data points represent the mean of concentrations in corresponding sampling intervals (n=10; Pearson test, p=0.0122, alpha=0.05).

3. The effect of physostigmine or atropine on lamotrigineinduced GABA response in cerebrospinal fluid dialysates
When cholinomimetic physostigmine was injected intraperitoneally at a dose of 0.5 mg/kg, a tendency to decrease in the GABA level of [0-40]-min sample was observed, but this did not yield a statistically significant difference (p =0.252). Nonselective muscarinic antagonist atropine (2 mg/kg) also produced non-significant increases in GABA [0-40]-min sample after atropine sulfate injection. Likewise, this increase did not yield a statistically significant difference (p=0.1023).

In order to analyze the involvement of cholinergic system in the lamotrigine induced GABA response, lamotrigine injections were given to physostigmine-pretreated rats (n=6). The percent maximum effects were 78.4 ± 10.2 and 74.7 ± 7.2 in physiological saline- and physostigmine- pretreated groups, respectively. Comparison of these data did not produce a statistical significant difference (p=0.8749).

Another group of rats received atropine sulfate pretreatment before lamotrigine injection (n=6) and the percent maximum GABA response was calculated as 85.5 ± 13.1. No statistical significant difference was found between physiological salineand atropine sulfate- pretreated groups (p=0.6354).

Results