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.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.
Methods
1. Animals and laboratoryWistar 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.
Results
1. The effect of lamotrigine injection on GABA and L-glutamic acid in cerebrospinal fluid dialysatesThe 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).
Reference
1) Bazil CW. New antiepileptic drugs. Neurology, 8:71–81, 2002.
2) Kwan P, Sills GJ, Brodie MJ. The mechanism of action of commonly
used antiepileptic drugs. Pharmacol Ther, 90:21–34,
2001.
3) Deckers CLP, Genton P, Sills GJ, Schmidt D. Current limitations
of antiepileptic drug therapy: a conference review. Epilepsy
Res, 53:1–17, 2003.
4) Ferrie CD, Panayiotopoulos CP: Therapeutic interaction of
lamotrigine and sodium valproate in intractable myoclonic
epilepsy. Seizure, 3:157–159, 1994.
5) Ferrie CD, Robinson RO, Knott C, Panayiotopoulos CP.
Lamotrigine as an add-on drug in typical absence seizures.
Acta Neurol Scand, 91:200–202, 1995.
6) Panayiotopoulos CP, CD Ferrie, C Knott, RO Robinson: Interaction
of lamotrigine with sodium valproate. Lancet, 41:445,
1993.
7) Pisani F, Di Perri R, Perucca E, Richens A. Interaction of lamotrigine
with sodium valproate. Lancet, 341:1224,1993.
8) Pisani F, Oteri G, Russo MF, Di Perri R, Perucca R, Richens A.
The efficacy of valproatelamotrigine comedication in refactory
complex partial seizures: evidence for a pharmacodynamic
interaction. Epilepsia, 40:1141–1146, 1999.
9) Messenheimer JA. Lamotrigine. Epilepsia, 36:87–94, 1995.
10) Calabrese JR, Bowden CL, Sachs GS, Ascher JA, Monaghan E,
Rudd GD. A double-blind placebo-controlled study of lamotrigine
monotherapy in outpatients with bipolar I depression.
Lamictal 602 Study Group. J Clin Psychiatry, 60:79–88, 1999.
11) Calabrese JR, Suppes T, Bowden CL, Sachs GS, Swann AC,
McElroy SL, et al.: A double-blind, placebo-controlled,
prophylaxis study of lamotrigine in rapid-cycling bipolar
disorder. Lamictal 614 Study Group. J Clin Psychiatry,
61:841–850, 2000.
12) Frye MA, Ketter TA, Kimbrell TA, Dunn RT, Speer AM, Osuch
EA et al.: A placebo-controlled study of lamotrigine and
gabapentin monotherapy in refractory mood disorders. J
Clin Psychopharmacol, 20:607–614,2000.
13) Bowden CL, Calabrese JR, Sachs GS, Yatham LN, Asghar SA,
Montgomery P, et al. A placebo-controlled 18-month trial of
lamotrigine and lithium maintenance treatment in recently
manic or hypomanic patients with bipolar disorder. Arch
Gen Psychiatry, 60:392-400, 2003.
14) Eriksson AS, O'Connor WT. Analysis of cerebrospinal fluid
amino acids in young patients with generalised refractory
epilepsy during an add-on study with lamotrigine. Epilepsy
Res, 34:75–83, 1999.
15) Kuzniecky R, Ho S, Pan J, Martin R, Gilliam F, Faught E, et
al. Modulation of cerebral GABA by topiramate, lamotrigine,
and gabapentin in healthy adults. Neurology, 58:368–372,
2002.
16) Lees G, Leach MJ. Studies on the mechanism of action of the
novel anticonvulsant lamotrigine (Lamactil) using primary
neurological cultures from rat cortex. Brain Res, 612:190–199,
1993.
17) Waldmeier PC, Baumann PA, Wick P, Feldtrauer JJ, Stierlin
C, Scmutz M. Similar potency of carbamazepine, oxcarbazepine
and lamotrigine in inhibiting the release of glutamate
and other neurotransmitters. Neurology, 45:1907–1913,
1995.
18) Waldmeier PC, Martin P, Stocklin K, Portet C, Scmutz M. Effect
of carbamazepine, oxcarbazepine and lamotrigine on the
increase in extracellular glutamate elicited by veratridine in
rat cortex and striatum. Naunyn-Schmiedeberg's Arch Pharmacol,
354:164–172, 1996.
19) Wang SJ, Sibra TS, Gean PW. Lamotrigine inhibition of glutamate
release from isolated cerebrocortical nerve terminals
(synaptosomes) by suppression of voltage-activated calcium
channel activity. NeuroReport, 2:2255–2258, 2001.
20) Cunningham MO, Jones RSG. The anticonvulsant, lamotrigine
decreases spontaneous glutamate release but increases
spontaneous GABA release in the rat entorhinal cortex in
vivo. Neuropharmacology, 39:2139–2141, 2000.
21) Cunningham MO, Wood SJ, Dhillon A, Jones RSG. Reciprocal
modulation of glutamate and GABA release may underlie the
anticonvulsant effect of phenytoin. Neuroscience, 95:343–351,
2000.
22) Braga MF, Aroniadou-Anderjaska V, Post RM, Li H. Lamotrigine
reduces spontaneous and evoked GABAA receptormediated
synaptic transmission in the basolateral amygdala:
implications for its effects in seizure and affective disorders.
Neuropharmacology, 42:522–529, 2002.
23) Hassel B, Tauboll E, Gjerstad L. Chronic lamotrigine treatment
increases rat hippocampal GABA shunt activity and elevates
cerebral taurine levels. Epilepsy Res, 43:153–163, 2001.
24) Tanganelli S, Bianchi C, Beani L. The modulation of cortical
acetylcholine release by GABA, GABAlike drugs and benzodiazepines
in freely moving guineapigs. Neuropharmacology,
24:291–299, 1985.
25) Bianchi C, Tanganelli S, Marzola G, Beani L. GABA induced
changes in acetylcholine release from slices of guinea-pig
brain. Naunyn Schmiedebergs Arch Pharmacol, 318:253–258,
1982.
26) McCormick DA, Prince DA. Mechanisms of action of acetylcholine
in the guinea pig cerebral cortex in vitro. J Physiol,
375:169–194, 1986.
27) Raiteri M, Marchi M, Paudice P, Pittaluga A. Muscarinic receptors
mediating inhibition of y-aminobutyric acid release in
rat corpus striatum and their pharmacological characterization.
J Pharmacol Exp Ther, 254:496–501, 1990.
28) Hasuo H, Gallager JP, Shinnick-Gallager P. Disinhibition in
the rat septum mediated by M1 muscarinic receptors. Brain
Res, 438:323-327, 1988.
29) Shirakawa J, Taniyama K, lwai S, Tanaka C. Regulation [3H]
GABA release from strips of guinea pig urinary bladder. Am
J Physiol, 255:888-893, 1988.
30) Yananli H, Gören MZ, Berkman K, Aricioğlu F. Effect of agmatine
on brain L-citrulline production during morphine
withdrawal in rats: a microdialysis study in nucleus accumbens.
Brain Res, 1132:51-58, 2007.
31) Paxinos G, Watson C. The rat brain in stereotaxic coordinates,
ed 2. London Academic Press 1986.
32) Yananli HR, Terzioğlu B, Goren MZ, Aker RG, Aypak C, Onat
FY. Extracellular hypothalamic gamma-aminobutyric acid
(GABA) and L-glutamic acid concentrations in response to
bicuculline in a genetic absence epilepsy rat model. J Pharmacol
Sci, 106:301-309, 2008.
33) PL Wheatley, AA Miller. Effects of lamotrigine on electrically
induced afterdischarge duration in anaes. thetised rat, dog,
and marmoset. Epilepsia, 30:34–40, 1989.
34) Morris RG, Black AB, Harris AL, Batty AB, Sallustio BC.
Lamotrigine and therapeutic drug monitoring: retrospective
survey following the introduction of a routine service. Br J
Clin Pharmacol, 46:547–551, 1998.
35) Walker MC, Tong X, Perry H, Alavijeh MS, Patsalos PN.
Comparison of serum, cerebrospinal fluid and brain extracellular
fluid pharmacokinetics of lamotrigine. Br J Pharmacol,
130:242–248, 2000.
36) Walton NY, Jaing Q, Hyun B, Treiman DM. Lamotrigine vs.
phenytoin for treatment of status epilepticus: comparison in
an experimental model. Epilepsy Res, 24:19–28, 1996.
37) Parsons DN, Dickins M, Morley TJ. Lamotrigine: absorption,
distribution, and excretion. In: Levy, R.H., Mattson, R.H.,
Meldrum, B.S. Antiepileptic Drugs, Raven Press, New York,
1995. pp 877– 881.
38) Leach MJ, Marden CM, Miller AA. Pharmacological studies
on lamotrigine, a novel potential antiepileptic drug: II. Neurochemical
studies on the mechanism of action. Epilepsia,
27:490–497, 1986.
39) Cheung H, Kamp D, Harris E. An in vitro investigation of the
action of lamotrigine on neuronal voltage-activated sodium
channels. Epilepsy Res, 13:107–112, 1992.
40) Lang DG, Wang CM, Cooper BR. Lamotrigine, phenytoin and
carbamazepine interactions on the sodium current present in
N4TG1 mouse neuroblastoma cells. J Pharmacol Exp Ther,
266:829–835, 1993.
41) Xie X, Lancaster B, Peakman T, Garthwaite J. Interaction of
the antiepileptic drug lamotrigine with recombinant rat brain
type IIA Na channels and with native Na channels in rat hippocampal
neurones. Pflugers Arch, 430:437–446, 1995.
42) Lizasoain I, Knowles RG, Moncada S. Inhibition by lamotrigine
of the generation of nitric oxide in rat forebrain slices. J
Neurochem, 64:636–642, 1995.
43) Mikati MA, Holmes GL. Lamotrigine in absence and primary
generalized epilepsies. J Child Neurol. Suppl, 1:29-37, 1997.
44) Ahmad S, Fowler LJ, Whitton PS. Effects of acute and chronic
lamotrigine treatment on basal and stimulated extracellular
amino acids in the hippocampus of freely moving rats. Brain
Res, 1029:41-47, 2004.
45) Van Der Zee EA, Luiten PGM. Cholinergic and GABAergic
neurons in the rat medial septum express muscarinic acetylcholine
receptors. Brain Res, 652:263–268, 1994.
46) De Boer P, Westerink BCH. GABAergic modulation of striatal
cholinergic interneurons: An in vivo microdialysis study.
J Neurochem, 62:70–75, 1994.
47) Tellioglu T, Akin S, Ozkutlu U, Oktay S, Onat F. The role of
brain acetylcholine in GABAA receptor antagonist-induced
blood-pressure changes in rat. Eur J Pharmacol, 317:301-307,
1996.
48) Onat F, Tellioglu T, Aker R, Goren Z, Iskender E, Oktay S. Effect
of muscimol on cholinomimetic-induced cardiovascular
responses in rats. Eur J Pharmacol, 362:173-181, 1988.
49) Kayadjanian N, Menetrey A, Besson MJ. Activation of muscarinic
receptors stimulates GABA release in the rat globus
pallidus. Synapse, 26:131–139, 1997.