Metabotropic glutamate receptor 1 alpha modulation of soman induced status epilepticus

METABOTROPIC GLUTAMATE RECEPTOR 1α : MODULATION OF SOMAN- INDUCED STATUS EPILEPTICUS LEONG AI LIN B.Sc, The University of Melbourne A THESIS SUBMITTED FOR THE DEGREE OF MASTERS OF SCIENCE DEPARTMENT OF PHARMACOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2003 ACKNOWLEDGEMENT “Great wisdom is lessons learnt from many men’s acts of foolishness.” We learn everyday and sad to say usually through mistakes In the pursuit of this MSc degree, I made mistakes, I corrected them and most importantly I learnt All these would be impossible if not for Assoc Professor Sim Meng Kwoon and Dr Tang Feng Ru Many thanks for their expert guidance, patient teachings and kind understanding I am very grateful to all the staff of DSO National Laboratories with whom I spent my 2½ years Many thanks especially to Mr Loke Weng Keong for giving me the chance to work in DSO and providing me his ever- ready- help in many areas of my study I would like to thank members of the Pharmacology and Toxicology Group, Emily, Doris, Yong Teng, Alicia and ex-members, Cynthia and Jing Ping who made my life in the group very happy and memorable Last but not least, my sincere thanks to my family and friends for all their support during the ‘darkest days’ of my life i TABLE OF CONTENTS Page List of Figures iv List of Tables v List of Abbreviations vi Summary of Thesis viii Chapter 1: Introduction Chapter 2: The Animal Model 2.1 2.2 2.3 Materials and Methods 2.1.1 Animals 2.1.2 Recordings of electroencephalogram 2.1.3 Intoxication by soman 2.1.4 Perfusion and cell histology 2.1.5 Neuropathology assessment 2.2.1 Seizure and EEG recordings 2.2.2 Histology Examination Results Discussion 15 Chapter 3: Metabotropic Glutamate Receptors Expression during SISE 3.1 Overview 3.2 Materials and Methods 3.2.1 3.3 Immunohistology staining 22 Effect of SISE on mGluR1α expression 23 Results 3.3.1 3.4 18 Discussion 26 Chapter 4: Post Treatment of SISE 4.1 Overview 4.2 Materials and Methods 28 ii 4.3 4.4 4.2.1 Drugs 32 4.2.2 Intracereboventricular (icv) administration of mGluR1 antagonist 32 4.3.1 Neuroprotective activity of diazepam 34 4.3.2 Neuroprotective activity of LY367385 36 Results Discussion 39 Chapter 5: Neurotransmitters and SISE 5.1 Overview 5.2 Materials and Methods 5.2.1 5.3 42 Microdialysis fiber implantation and perfusion 46 5.2.2 Analysis of amino acids 47 5.2.3 Statistical analysis 47 5.3.1 Chromatographic separation 48 5.3.2 Effects of LY367385 on extracellular Results glutamate and GABA 5.3.2.1 Delivery via icv 5.3.2.2 Delivery via reverse microdialysis 5.4 Discussion 50 52 56 References 60 Appendix A A-1 iii LIST OF FIGURES Figure Title Page 2.1 Phases of electroencephalogram pattern changes observed in rats intoxicated with soman 10 2.2 Cresyl violet staining at piriform cortex of vehicle (saline)treated rat, and soman- treated rats 11 2.3 Pathology in the piriform cortex day after soman-induced SE visualized by cresyl violet stain 12 2.4 Cell quantification at layer II and III of the piriform cortex at different time intervals after soman intoxication 14 3.1 Immunoreactivity for mGluR1α in layer III of the piriform cortex 24 3.2 mGluR1α immunoreactivity in the piriform cortex at different time intervals after soman intoxication 25 4.1 Chemical structure of LY367385 31 4.2 Neuroprotective effect of diazepam administrated intramuscularly 20 minutes post seizure onset 35 4.3 Cresyl violet staining at piriform cortex of SISE rats treated with combinations of LY367385 and diazepam 38 5.1 Resolution and detection of glutamate and GABA by HPLC 49 5.2 Time course of changes in glutamate output induced by SISE and SISE + post- treatment with LY367385 and diazepam 51 5.3 Effects of reverse microdialysis of LY367385 on the release of glutamate and GABA from the piriform cortex 54 5.4 Time course of changes in glutamate and GABA output induced by reverse microdialysis of 5mM LY367385 in the piriform cortex of SISE rats 55 iv LIST OF TABLES Table Title Page 4.1 Neuroprotective effect of drug combination treatment on SISE 37 5.1 Pathways utilizing glutamate as a transmitter 43 v LIST OF ABBREVIATIONS 2-PAM Pralidoxime-2-chloride 3HPG 3-hydroxyphenylglycine AC Adenyl cyclase ACh Acetylcholine AChE Acetylcholinesterase ACSF Artificial cerebo- spinal fluid AIDA (RS)-1-aminoindan-1,5-dicarboxylic acid AMN Atropine methyl nitrate AMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid BSA Bovine serum albumin CA Cornu ammonis CBPG (S)-(+)-2-(3’-carboxybicyclo[1.1.1]pentyl)- glycine ChE Cholinesterase CV Cresyl violet DHPG 3, dihydroxyphenylglycine EAA Excitatory amino acid ED50 50% Effective dose EEG Electroencephalogram EPSP Excitatory postsynaptic potential GA Tabun GABA γ- aminobutyric acid GB Sarin GD Soman GTP Guanine triphosphate HPLC High pressure liquid chromatography i.m Intramuscular i.p intraperitoneally IAA Inhibitory amino acid icv Intracerebroventricular iGluR ionotropic glutamate receptor IOD integrated optical density IP3 inositol-1,4,5-trisphosphate LD50 50% lethal dosage vi LY367385 (+)-2-methyl-4-carboxyphenylglycine mGluR Metabotropic glutamate receptor NMDA N-methyl-D-aspartate OD optical density OP Oraganophosphorus OPA O-phthalaldehyde PB Phosphate buffer PBS Phosphate buffered saline PBSTx Phosphate buffered saline with 0.1% Triton X PI Polyphosphoinositide PKC Protein kinase C PLC Phospholipase C sc Subcutaneous SE Status epilepticus SISE Soman-induced status epilepticus TBS Tris- buffered saline VX O-ethyl-S-[2-(diisopropylamino)ethyl] methylphosphonothioate vii SUMMARY OF THESIS Soman (pinacolyl methylphosphonofluoridate), a potent irreversible acetylcholinesterase (AChE) inhibitor, induces status epilepticus (SISE) in rats during severe intoxication Subsequent neurodegeneration in the limbic structures is a result of hyperexcitability of the epileptic brain and is thought to be mediated by activation of glutamate receptors The aim of this study was to focus on the role played by metabotropic glutamate receptor (mGluR) and the effect of selective blockage of mGluR1α as a post- treatment of SISE In this current work, an initial effort was concentrated on the establishment of an in vivo animal model to facilitate the study of post-SISE treatment drugs An intoxication regime was drawn up to include a pre-treatment combination of pyridostigmine bromide and atropine methyl nitrate (AMN) administered 30 minutes prior to a convulsive dose of 176µg/ kg (equivalent to 1.6LD50 ) soman injected subcutaneously into Wistar rats, followed by injection of pralidoxime-2-chloride (2-PAM) and an additional dose of AMN Pathological symptoms such as mastication, motor convulsion and salivation were exhibited within minutes of exposure A histological examination of the brain sections revealed a progressive neuronal loss throughout a one- month time course after intoxication Piriform cortex, amygdala and hippocampus are the most vulnerable structures Immunohistological staining for mGluR1α in the piriform cortex showed a 3-fold upregulation of expression from basal level at hour post-SISE Activation of Group I mGluR produces long-lasting epileptiform discharges in the hippocampus (Merlin and Wong, 1997) and intracerebroventricular (icv) injection of 3, 5-dihydroxyphenylglycine (DHPG) into rats induced seizure and subsequent neuronal viii damage (Camon et al., 1998) To investigate if such an activation of mGluR1α in our SISE model is neurotoxic, a selective mGLuR1α antagonist, LY367385, was used Icv administration of 4- 400 nmole of LY367385 at 15 minutes after seizure onset did not terminate seizure activity nor showed any neuroprotection Cell density, regardless of dosage tested, was not significantly different from that of soman-intoxicated untreated animals In another experiment, a combination of diazepam and LY367385 was tested LY367385 (200 nmole, icv) was given at 15 minutes after seizure onset, followed by intramuscular injection of mg/kg diazepam minutes later Though seizure termination was not observed in this group, such a combination improved the percentage of cell survival in layer II and III of the piriform cortex by more than 2.5 and 3.5 times, respectively, as compared to untreated rats or rats that received only diazepam (2 mg/kg) treatment At a higher dose of combination - 400 nmole of LY367385 and mg/kg of diazepam, three out of six rats displayed lowered amplitude EEG spikes within 20 - 30 minutes after drug treatment and seizure activity was altogether terminated by the fourth hour These three animals did not suffer any neuropathology and their cell density in the piriform remained unchanged from the control level In a subsequent study, reverse microdialysis of mM LY367385 solution into the piriform cortex during SISE increased the release of the inhibitory amino acid GABA by 300- fold and reduced the excitatory amino acid glutamate to 10% of basal level However, this mode of local administration to the piriform cortex was not effective in ix The number of cells in the piriform cortex was determined as previously described in Section 4.3 to be 679 ± 33 (mean ± SEM) cells/ mm2 at Layer II and 246 ± 76 cells/ mm2 at Layer III These cell densities in Layer II and III of the piriform cortex were 3folds and at least 7- folds, respectively, more than those observations made in untreated SISE rats (Table 4.1) Albeit, complete neuroprotection was not conferred, these values represent close to 70% and 40% of Layer II and III, respectively, of the piriform cortex observed in non- intoxicated rats 53 Glutamate 160% 140% 120% 100% 80% 60% 40% Amino Acid output (% of mean basal level) 20% 0% 15 30 45 60 45 60 GABA 7000% 6000% 5000% 4000% 3000% 2000% 1000% 0% 15 30 Time in Fig.5.3 Effects of reverse microdialysis of LY367385 on the release of glutamate (upper graph) and GABA (lower graph) from the piriform cortex Value at time represents mean of basal dialysates Treatment duration is indicated by a black bar Solid line ( ) traced neurotransmitter level in rats administered with 1mM LY367385 and broken line ( ) 5mM 54 Glutamate 120% 100% 80% * 60% 40% * 45 60 * 20% Amino Acid output % of mean basal * 0% 15 30 75 90 105 120 135 150 165 Soman intoxication 45000% * 40000% GABA 35000% 30000% 25000% * 20000% 15000% 10000% 5000% 0% * 15 Soman intoxication 30 45 60 75 Time in Fig 5.4 Time course of changes in glutamate (upper graph) and GABA (lower graph) output induced by reverse microdialysis of 5mM LY367385 in the piriform cortex of SISE rats Value at time represents mean of basal dialysates Treatment duration as marked by black bar *p[...]... potent glutamate receptor agonist, damaged limbic structures by inducing limbic seizures in rats (Lothman and Collins, 19 81; BenAri et al., 19 81) This finding spurred a series of extensive studies on glutamate excitotoxicity leading to the hypothesis that excessive amounts of glutamate were 18 released during limbic seizures, causing cell damage (Wade et al., 19 87; Lallement et al., 19 91) The glutamate. .. and neurotoxic (see review in Bruno et al., 20 01) Moreover expression of mGluR1α was implicated to play a neurotoxicity role in the hippocampus of rat pilocarpine model of status epilepticus (Tang et al., 20 01) 20 Therefore, in this chapter, the investigation will focus on the effect of SISE on the expression of mGluR1α 21 3.2 Materials and Methods 3.2 .1 Immunohistology staining The free-floating sections... profound ataxia (Haggerty and Brown 19 96) While studies on iGluR are underway, a new class of glutamate receptors called the metabotropic receptors (mGluRs) was discovered in the 19 80s Unlike iGluR, mGluR 19 is coupled to GTP-binding protein (G protein) mGluR is linked to the activation of phospholipase C (PLC) and inhibition or activation of adenylyl cyclases (AC) (see review in Bruno et al., 20 01) ... in layer III of the piriform cortex of (A) a saline-vehicle treated rat and (B) a rat after 8 hours of SISE Note the heavily immunolabelled cell bodies and dendrites in (B), implying an increased expression of mGluR1α induced by SISE 24 350 * 300 250 IOD 200 * * 15 0 10 0 50 0 c o n tr o l h8 d1 w1 w4 Fig 3.2 mGluR1α immunoreactivity in piriform cortex at different time intervals after soman- intoxication... intoxication dosage of soman was chosen to be 1. 6LD50 (17 6 mg/kg) based on a similar study by McDonough and Shih (19 97) The choice of intoxication dosage was critical as a dose less than 0.8LD50 rarely induced clinically- displayed seizure symptoms (Baille et al., 20 01) On the other hand, at doses > 1. 8LD50, animals developed generalized seizures and mortality was high (Shih et al., 19 91) The chosen intoxication... antagonists at the glutamate receptors can bring about facilitation or inhibition of excitatory postsynaptic potentials (EPSP) The aim of this study was to focus on the role played by metabotropic glutamate receptor (mGluR) and the effect of selective blockage of mGluR1α as a post- treatment of SISE The results demonstrate that neuropathology could be detected as early as 8 hours of SISE At the same time-... animal model, a dose of 1. 6LD50 (17 6µg/kg) was adopted The intoxication method used was similar to Ballough et al., (19 95) A pretreatment regimen was included to reduce the acute lethality An intramuscular (i.m.) dose of 0 .13 mg/kg of pyridostigmine bromide and either 4mg/kg or 8mg/kg of AMN were given 30 minutes before soman administration Immediately following soman, an i.m injection of pralidoxime-2-chloride... than 30 min (C) Fig.2 .1 Phases of electroencephalogram pattern changes observed in rats intoxicated with soman (A) Baseline EEG was obtained before receiving pretreatment and soman insult (B) 10 minutes after soman administration, high amplitude rhythmic spikes marked the onset of seizure (C) Stable trains of high amplitude spikes interrupted by flat periods during status epilepticus 10 A B III II I C... condensed nucleus 12 Cell quantification to show progressive neuronal loss in the piriform cortex throughout the one-month time-course was shown in Fig 2.4 The most pronounced decrease was found on day 1 after soman insult, as cell density in layer III fell to half of that of controls By week 4, remaining cells were less than 30% of normal cell density 13 16 % 14 % Mean cell density 12 % * * 10 % * * * 8% *... chapters 17 Chapter 3 Metabotropic Glutamate Receptors Expression during SISE 3 .1 Overview As seen in Chapter 2, the pathological abnormalities are exhibited by soman- poisoned subjects within minutes of exposure and neuropathology could be detected 8 hours after seizure onset Hence, early arrest of seizure is an important strategy in overcoming the neurotoxicity caused by soman Since the hyperexcitation of ... half of that of controls By week 4, remaining cells were less than 30% of normal cell density 13 16 % 14 % Mean cell density 12 % * * 10 % * * * 8% * * 6% * 4% 2% 0% control n=3 h8 d1 n=6 n=6 w1 n=4... Materials and Methods 2 .1. 1 Animals 2 .1. 2 Recordings of electroencephalogram 2 .1. 3 Intoxication by soman 2 .1. 4 Perfusion and cell histology 2 .1. 5 Neuropathology assessment 2.2 .1 Seizure and EEG recordings... Discussion 15 Chapter 3: Metabotropic Glutamate Receptors Expression during SISE 3 .1 Overview 3.2 Materials and Methods 3.2 .1 3.3 Immunohistology staining 22 Effect of SISE on mGluR1α expression