AMINO ACID EFFECTS IN THE FELINE CENTRAL NERVOUS SYSTEM Martin James Peet A thesis submitted for the Degree of Doctor of Philosophy in the Au5trali an National University. November, 1982 Due to the complex nature of the neuropharmacological experiments described in this thesis, collaboration with Professor D.R. Curtis (Chapters 3 and 7) and other members of the Department of Pharmacology was necessary. Dr J.D. Leah participated in the experiments described in Chapters 3,4,5 and 7, Dr J.C. Bornstein in those described in Chapter 7 and Mr R. Malik in the experiments of Chapter 6. ' . (Martin James Peet). . ' ·:. . ' . ........l. ~ . ' ·•.· . '. •-t\," ~ . """' ·-v,,,:rC:t, :.~. ·' !•~ oJ ~, :,. ", '-:!-"" ... ''"' • ·.. ;zt .. .,..:tc-.. ~··,•tr During the tenure of my Australian National University Scholarship the following papers have been published or accepted for publication: Curtis, D.R., Leah, J.D., Peet, M.J. Lack of specificity of a 'taurine' antagonist. Brain Research 244 (1982) 198-199. Curtis, D.R., Leah, J.D. and Peet, M.J. Effects of noradrenaline and 5-hydroxytryptamine on spinal Ia afferent terminations. Brain Research (1982) Curtis, D.R., Lodge, D., Bornstein, J.C. and Peet, M.J. Selective effects of (-)-baclofen on spinal synaptic transmission in the cat. Exp. Brain Res. 42 (1981) 158- 170. Curtis, D.R., Lodge, D., Bornstein, J.C., Peet, M.J. and Leah, J.D. The dual effects of GABA and related amino acids on the electrical threshold of ventral horn group Ia afferent terminations in the cat. Exp. Brain Res., (1982) Krogsgaard-Larsen, P., Hansen, J.J., Lauridsen, J., Peet, M.J., Leah, J.D. and Curtis, D.R. Glutamic acid agonists. Stereochernical and conformational studies of DL-a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and related compounds. Neurosci. Lett. 31 (1982) 313-317. Krogsgaard-Larsen, P., Hjeds, H., Curtis, D.R., Leah , J.D . and Peet, M.J. Glycine antagonists structurally related to muscimol, THIP and isoguvacine. J. Neurochem. , (1982) Ii 11 I Krogsgaard-Larsen, P., Mikkelsen, H., Jacobsen, P., Falch, E., Curtis, D.R., Peet, M.J., Leah, J.D. Thio-THIP and I related compounds. Synthesis and biological activity. J. I ' Med. Chem. (1983) ' I I Peet, M.J., Leah, J.D., Watkins, J.C. and Curtis, D.R. I ll Antagonists of the synaptic and amino acid excitation of neurones in the cat spinal cord. Brain Research II .(1982) • II Peet, M.J., Malik, R. and Curtis, D.R. Postexcitatory 'I depression of neuronal firing by acidic amino acids and acetylcholine in the cat spinal cord. Brain Research (1983) I Willow , M., Peet, M.J., Johnston, G.A.R. and Curtis , D.R . In vitro and in vivo effects of pentobarbitone sodium. J. Pharm. Pharmac., (1982) I II Ii II ltl [11 I Ii I I ,, I I' ! l II p 1, 11 11, llt ' TABLE OF CONTENTS Page No. ' ii ACKN'OWLEDGEMENTS ............................... . 1 ' S ~RY. • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 2 I , 1 • GENERAL INTRODUCTION ...................... . 4 I (a) Synthesis and storage within, and release from, presynaptic terminals. . . . . . . . . . . . . Synthesis and storage .. 5 Release . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 (b) Receptors for amino acids and their role in synaptically-induced postsynaptic events. Ligand binding studies 18 • • • • • • • • • • • • Microelectrophoretic studies ...... . 22 Rdle of amino acids in synaptic transmission...................... 32 11 : ' ~ (c) Interaction with postsynaptic receptors i I I and the underlying ionic mechanism(s) of ;I I I 1': action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 6 !I 11 I', I (d) Termination of the action of amino I I acids....... . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 II I t I 1(1 i, 2. METHODS I I (a) General methods ..................... . 57 I (b) Spinal cord preparation ............. . 58 II:' (c) Leg preparations ..................... 59 ,, (d) Microelectrophoretic techniques ...... 60 Ii I! I (e) Experimental design I ( 1 ) Chapter 3 •••••••••••• 62 C. 0 ••••••••• (2) Chapter 4 ...................... 64 0 (3) Chapter 5 .......•............... 65 I (4) Chapter 6 ............ 66 0 •••••••••• I ! (5) Chapter 7••••••••o••• • •o•••••••• 67 I I .l I Location and indentification of : Group Ia terminations . ..... 67 l e••••o I I i I .\ Afferent volley p AD .. 71 G ••••••••••• I r I I Measurement of coupling resistances 71 i i II t ii Microelectrophoretic methods . . o •• 72 \f I I I Sources of drugs and chemicals .. 74 o ' II II ' 3. GLUTAMIC ACID AGONISTS: STEREOCHEMICAL AND CONFORMATIONAL STUDIES OF AMPA AND RELATED COMPOUNDS Introduction ............................ . 75 ' i I 1' (, Results ................................. . 78 . . Discussion .............................. . 78 'i i 4. CLASSIFICATION OF RECEPTORS FOR EXCITANT AMINO ACIDS ·,r 'i I Pl Introduction.............................. 83 ll\ 1: Ii Results ............... .................. . 86 I! ~ II (a) Effect of 2,3PDA and 2,SPDA on amino 1, II Ii acid-induced excitation ........ , ... . 87 ii !1 Ii Ill (b) Dipeptide analogues as amino acid antagonists......................... 87 1 (c) The effects of phosphonate analogues 88 on amino acid-induced excitation .... . . D1.scuss1.on ................................. . 89 I i I : .I II Ii I ' ' : 5. AMINO ACIDS IN SYNAPTIC TRANSMISSION: USE OF ANTAGONISTS Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 4 Results................................. 96 (a) Effects 2,3PDA and 2,SPDA on synaptic excitation........................ 97 (b) Dipeptide analogues as antagonists of synaptic excitation............... 98 (c) The effects of phosphonate analogues on synaptic excitation............ 98 Discussion.............................. 99 6. POSTEXCITATORY DEPRESSION OF NEURONAL FIRING BY ACIDIC AMINO ACIDS AND ACETYLCHOLINE Introduction.......................... 102 Results............................... 103 (a) Amino acid and acetylcholine-evoked PED: effects of inhibitory amino acid antagonists....... . . . . . . . . . . . . . 103 (b) Reduction of amino acid or acetylcholine excitations by antagonists: effects on PED ............................ 105 II I (c) Changes in the amount or duration for ejection of excitants: effect I I on PED ........................ . 105 i ' i. scussi.o n ................................. . 106 D 7. THE EFFECTS OF GABA, RELATED AMINO ACIDS AND AMINES ON THE ELECTRICAL THRESHOLD OF VENTRAL HORN GROUP IA AFFERENT TERMINATIONS IN THE CAT Introduction................................ 111 Results..................................... 115 : (a) Effects of amino acids on termination : 11 thresholds..................... 115 Ii (b) The effects of amines on termination ' thresholds..................... 1 22 ' (c) The effect of amino acids and amines II 1, ii on coupling resistance......... 122 l (d) Ouabain: effects on amino acid-induced l 11 alterations in termination threshold l :: and coupling resistance. . . . . . . . 1 23 I: 1 ,, I 'i I! I i. scussi.o n ................... .... ... . ...... . 126 D I ' r, 11 I! BIBLIOGRAPHY ............................... . 132 :, I', I! 1 I I I I ACKNOWLEDGEMENTS i I I would like to extend my sincere thanks to I I Professor D.R. Curtis for the opportunity to study in the Department of Pharmacology and his supervision during the I tenure of my scholarship. The collaboration of Drs J.C. 1, Bornstein and J.D. Leah and Mr R. Malik, and the provision 1: of compounds by Drs. G.A.R. Johnston (The University of Sydney) G.G. Yarbrough (Merck Institute of Therapeutic Research, USA) and in particular P. Krogsgaard-Larsen (The Royal Danish School of Pharmacy) and J.C. Watkins (The ' I University of Bristol) is gratefully acknowledged. I am also grateful for the technical assistance provided by 1, I I members of the Department of Pharmacology, especially Mrs t P.J. Searle and Mrs M.E. Rodda. 11 Ii ii Ii ii .\ i ll II 1, 1, 1, I j I i,