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Analysis of excitatory amino acid receptors in the rat spinal cord in vivo and in vitro Magnuson, David Stuart Keith 1988

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ANALYSIS OF EXCITATORY AMINO ACID RECEPTORS IN T H E RAT S P I N A L C O R D IN V I V O A N D IN V I T R O by DAVID STUART KEITH  MAGNUSON  B . S c , University of Victoria, 1983 A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S FOR T H E D E G R E E O F DOCTOR OF  PHILOSOPHY in  THE FACULTY OF GRADUATE STUDIES  (Neuroscience)  W e accept this thesis as conforming to the required s t a n d a r d  T h e University of British C o l u m b i a April 1988 © David Stuart Keith M a g n u s o n , .1988  In  presenting  degree freely  at  this  the  available  copying  of  department publication  of  in  University  of  for  this or  thesis  this  British  reference and  thesis by  partial  for  his thesis  or for  her  DE-6(3/81)  1Y3  study.  Columbia  I  of I  further  purposes  gain  the  shall  requirements  agree  that  agree  may  representatives.  financial  Department  V6T  Columbia,  scholarly  permission.  The University of British 1956 Main Mall Vancouver, Canada  fulfilment  be  It not  is  that  the  Library  permission  granted  by  understood be  for  allowed  an  advanced  shall for  the that  without  make  it  extensive  head  of  my  copying  or  my  written  Abstract Several e n d o g e n o u s amino acids including L-glutamate a n d L-aspartate have potent excitatory effects in the central nervous system. They are thought to act as synaptic transmitters in many neural pathways including those in the spinal cord. Three distinct receptors have been described through which these excitatory amino acids exert their effects. These are referred to as quisqualate, kainate a n d N-methyl-D-aspartate (NMDA) receptors, after the e x o g e n o u s excitants most specific for each.  In addition, sub-types of the N M D A receptor have been  proposed to account for differences observed in the actions of the e n d o g e n o u s excitant quinolinate (2,3-pyridine dicarboxylate) in various regions of the nervous s y s t e m . T h e characterization of excitant amino acid receptors has been a c c o m p l i s h e d primarily using two or more potent antagonists which include D-(-)-2-amino-5-phosphonovalerate (APV), a specific N M D A antagonist, and kynurenate, a c o m p o u n d related to quinolinate which potently attenuates the actions of N M D A - a n d kainate-like excitants. Structure-activity studies of amino acid receptors were undertaken using standard extracellular recording and iontophoretic techniques in the dorsal horn of the spinal c o r d in vivo, and c o m p a r e d with the neocortex of the rat. In addition, a spinal cord slice preparation w a s d e v e l o p e d wherein dorso-ventral longitudinal slices were prepared from the lumbar enlargement of weanling rats (50 - 125 g). T h e slices were maintained in an "interface" tissue bath of novel design. Extracellular recording of several hours duration and up to 8 hours after slice - ii -  preparation w e r e routinely possible. Conformationally restricted analogues of glutamate, aspartate a n d quinolinate were e x a m i n e d for agonist and antagonist actions in the rat spinal cord in vivo and in vitro. C o m p o u n d s found to be excitants were c o m p a r e d directly with quisqualate, kainate, and N M D A for sensitivity to blockade by A P V and kynurenate applied both iontophoretically a n d in the bathing m e d i u m ; antagonist doseresponse curves were constructed for the actions of A P V and kynurenate against quisqualate, kainate, quinolinate a n d NMDA.  The conformationally restricted  c o m p o u n d s found to be antagonists were e x a m i n e d to determine their potency and specificity against excitations elicited by quisqualate, kainate, quinolinate and NMDA. Although quinolinate is known to be NMDA-like in the hippocampus and cortex, w h e n c o m p a r e d to quisqualate, kainate a n d N M D A in the spinal cord in vitro, it proved to be unique. A fourth receptor (the " Q U I N " receptor) is proposed to account for its actions in the spinal cord. Three of the isomers of 1-amino-1,3-cyclopentane dicarboxylate ( A C P D ) , conformationally restricted analogues of glutamate, were potently blocked by A P V and K Y N A and were therefore classified as NMDA-like. The fourth,  D-trans-ACPD.  w a s indistinguishable from quinolinate in terms of both potency and sensitivity to antagonists. The (-) isomer of trans-1-amino-1,2-cyclopentane dicarboxylate proved to be an antagonist with greater potency against excitations  - iii -  elicited by quisqualate and kainate than those of N M D A . T h e s e findings are, in many w a y s , different from what has been observed in the hippocampal slice. Several pyridine derivatives were e x a m i n e d ; 2,5- and 2,6-pyridine dicarboxylate were w e a k excitants behaving like quisqualate in the presence of A P V a n d kynurenate.  No other pyridines were excitatory; however 2,4-pyridine  dicarboxylate w a s observed to be a weak, non-specific antagonist similar in action to acridinate (an antagonist closely related to kynurenate). None of the pyridine derivatives, save quinolinate, are excitatory in the hippocampus. Structural analysis of the active c o m p o u n d s tested, in consideration of previous studies, shows that three points of attachment (two carboxyl and one amino group) are necessary for activation of N M D A , quisqualate and quinolinate receptors in the spinal cord. The location of the distal or y-carboxyl group relative to the a ionic groups appears to be the primary factor determining the activity of a conforrnationally restricted c o m p o u n d . The absolute distance b e t w e e n the Y-carboxyl a n d a-carbon appears to play a secondary role in determining the action of a c o m p o u n d .  -iv-  Table of Contents Abstract  ii.  List of Tables  viii.  List of Figures  x.  Acknowledgements  I.  xii.  Introduction A.  History of excitatory amino acid research  B.  Other e n d o g e n o u s excitatory amino acids a n d antagonists  10.  C.  Intracellular studies in the hippocampus and cortex  13.  D.  Excitatory amino acid receptor channels  14.  E.  Synaptic transmission in the spinal cord  16.  F.  Synaptic roles in the hippocampus and cortex  18.  G.  Actions of glutamate a n d aspartate  21.  H.  Quinolinate a n d other NMDA-like agonists  23.  C y c l o p e n t a n e a n d pyridine derivatives  24.  J.  Structure of the dorsal horn  25.  K.  In vitro slice preparations  26.  I.  II.  Aims of the present studies  III.  Methods  page  1.  28.  A.  Spinal cord and cortex in vivo  32.  B.  Spinal cord slice preparation  33.  C.  Maintaining spinal cord slices  38.  D.  Design of tissue c h a m b e r  39.  E.  Microiontophoresis  44.  F.  Recording electrical activity  45.  -v-  IV.  Results A.  Presentation of the d a t a  page 48.  B.  Archetypal agonists and antagonism by PDA, K Y N A and A C R A in the spinal cord in vivo  C.  Archetypal agonists and antagonism by PDA, K Y N A and A C R A in the cortex in vivo  D.  oc-Substituted analogues in the spinal cord in vivo  E.  (+) and M t r a n s - c v c l o p e n t a n e aspartate in the spinal cord in vivo  F.  56. 61. 61.  Archetypal agonists and antagonism by A P V , PDA, KYNA and A C R A in the spinal cord in vitro  G.  L- and D-glutamate in the spinal cord in vitro  H.  Q U I N in the spinal cord in vitro: antagonists applied iontophoretically a n d topically  I.  54.  65. 71. 74.  Conformationally restricted a n a l o g u e s : 1-amino-1,3-cyclopentane a n d pyridine dicarboxylates  82.  J.  C o m p o u n d s acting at N M D A receptors  83.  K.  C o m p o u n d s acting at Q U I S receptors  88.  L.  QUIN-like activity  92.  V.  Discussion A.  Interpretation of data and results  B.  Excitation of dorsal horn neurones by the archetypal agonists Q U I S , KAIN and N M D A in vivo and in vitro  C.  104.  The aromatic antagonists A C R A and KYNA in the spinal cord and cortex  F.  103.  A n t a g o n i s m of cortical neurone responses to amino acid agonists by K Y N A and A C R A in vivo  E.  101.  A n t a g o n i s m of dorsal horn neurone responses to archetypal agonists by PDA, KYNA, A C R A and A P V in vivo  D.  96.  105.  a-Substituted analogues of glutamate  - vi -  106.  G.  (+) a n d (-)Trans-1 - a m i n o - 1 . 2 - c v c l o p e n t a n e dicarboxylate in vivo  H.  page  Archetypal agonists and blockade of their actions in the spinal cord in vitro  I.  111.  Actions of D- and L-glutamate in the spinal cord in vitro  112.  J.  Does quinolinate act at a fourth receptor?  113.  K.  The actions of the isomers of A C P D a n d several pyridine derivatives related to quinolinate A n a l o g u e s acting at spinal cord N M D A receptors  117.  M.  Quinolinate-like c o m p o u n d s  118.  N.  Quisqualate-like c o m p o u n d s  119.  O.  Regional differences in amino acid structure-activity  VI.  121.  Structure-activity relationships, model analysis A.  NMDA-like c o m p o u n d s  123.  B.  QUIN-like c o m p o u n d s  126.  C.  QUIS-like c o m p o u n d s  126.  D.  Other conformationally restricted c o m o u n d s  130.  Contributions made to the study of the spinal cord and excitatory amino acids in the central nervous system  VIII. IX.  116.-  L.  relationships  VII.  107.  132.  Structure-activity conclusions  134.  References  138.  - vii -  List of Tables Table 1.  Full names and abbreviations of all c o m p o u n d s tested.  Table 2.  Data from spinal cord in vivo: % reduction and X / N M D A for Q U I S , KAIN and N M D A vs. PDA, KYNA and A C R A .  52.  Data from spinal cord in vivo: ranked order of paired d a t a for L-GLU, QUIS, KAIN and N M D A vs. PDA, K Y N A and A C R A .  53.  Data from cortex in vivo: % reduction and X / N M D A for Q U I S , Q U I N , KAIN and N M D A vs. K Y N A and A C R A .  59.  Data from cortex in vivo: ranked order of paired d a t a for Q U I S , Q U I N , KAIN a n d N M D A vs. K Y N A and A C R A .  60.  Table 3.  Table 4.  Table 5.  Table 6.  page 30.  Data from spinal cord in vivo: % reduction for L-GLU, A M P A , Q U I S , KAIN and N M D A vs. a p C P G and a p F P G .  Table 7.  Data from spinal cord in vivo: ranked order of paired d a t a for L-GLU, A M P A , Q U I S , KAIN and N M D A vs. a p F P G .  Table 8.  Table 9.  62.  62.  Data from spinal cord in vivo: % reduction for Q U I S , L-GLU, KAIN and N M D A vs. (-) and (+)trans-CPA.  66.  Data from spinal cord in vivo: ranked order of paired d a t a for Q U I S , L-GLU, KAIN and N M D A vs. M t r a n s - C P A .  66.  Table 10. D a t a from spinal cord in vivo and in vitro: X / N M D A for Q U I S , Q U I N and KAIN vs. KYNA, A C R A , PDA a n d APV.  68.  Table 1 1 . C o m p a r i s o n of agonist a n d antagonist iontophoretic ejection currents used in vivo and in vitro.  70.  Table 12. Data from spinal cord in vitro: % reduction, X / N M D A a n d agonist potency for D-GLU, L-GLU and QUIS vs. APV a n d KYNA.  72.  Table 13. D a t a from spinal cord in vitro: ranked order of paired d a t a for Q U I S , L-GLU, D-GLU and N M D A vs. APV.  73.  Table 14. Data from spinal cord in vitro: % reduction and X / N M D A ratio for Q U I S , Q U I N , KAIN and N M D A vs. APV, KYNA and ACRA.  76.  - viii -  Table 15. Action of perfusate applied antagonists: IC50's  page  79.  Table 16. Data from spinal cord in vitro: ranked order of paired d a t a for Q U I S , Q I I I N , KAIN and N M D A vs. APV, K Y N A and A C R A .  81.  Table 17. D a t a from spinal cord in vitro: effects of M g - f r e e bathing medium on responses to Q U I S , Q U I N a n d N M D A .  82.  2+  Table 18. D a t a from spinal cord in vitro: % reduction, X / N M D A and agonist potency for 2,5- a n d 2,6-PyrDA, Q U I S , L-GLU, Q U I N , D-trans-. L-cjs-, L-trans- and  D-cis-ACPD a n d NMDA vs. APV.  85.  Table 19. Data from spinal cord in vitro: ranked order of paired d a t a for Q U I S , Q U I N , D-trans-. L-cjs.-, L-trans- and D-cjs_-ACPD and N M D A vs. A P V and KYNA.  87.  Table 20. D a t a from spinal cord in vitro: % reduction and X / N M D A for 2,5- and 2,6-PyrDA, Q U I S , L-GLU, Q U I N , D-trans-. L-cjs.-, L-trans- a n d D-cjs.-ACPD a n d N M D A vs. KYNA.  91.  Table 2 1 . Data from spinal cord in vitro: ranked order of paired d a t a for Q U I S , 2,5- and 2,6-PyrDA and N M D A vs. APV and KYNA.  93.  Table 22. B o n d lengths used with the Orbit Molecular Building S y s t e m .  124.  Table 23. Interionic distances determined by analysis of scale models.  125.  Table 24. Interionic distances of other analogues.  129.  Table 25. Interionic distances of conformationally restricted antagonists  130.  - ix -  List of Figures Figure 1.  Structures of: aspartate, glutamate, quisqualate, kainate, N-methyl-D-aspartate, a-methylglutamate, a-(para-fluoro)phenylglutamate.  Figure 2.  Figure 3.  Figure 4.  Figure 5.  Figure 6.  Figure 7.  page 3.  Structures of: D-cis-. D-trans-. L-cis- and L-trans-ACPD: (+) and (-)lrans-CPA, and (±)cJ£-2,3-PDA.  8.  Structures of: Q U I N , phthalate, picolinate, 3-hydroxypicolinate, A C R A , KYNA, 2,4-, 2,5- and 2,6-PyrDA.  12.  Diagrams of: the lumbar enlargement of the rat spinal cord in cross section; a single longitudinal slice.  36.  Diagram of: side and top views of a single c h a m b e r tissue bath of conventional design.  40.  Diagram of: side and top views of a t w o - c h a m b e r e d tissue bath of novel design.  41.  Bar graph showing the rise and fall in concentration of tryptamine added to the superfusate.  43.  Figure 8.  Diagram of the experimental apparatus.  46.  Figure 9.  Ratemeter records; spinal cord in vivo: A. Q U I S , L-GLU and N M D A vs. PDA a n d B. Q U I S , KAIN and N M D A vs. H t r a n s - C P A and KYNA.  50.  Figure 10.  Figure 1 1 .  Figure 12.  Figure 13.  Ratemeter records; cortex in vivo: A. QUIS and N M D A vs. KYNA and B. Q U I N and N M D A vs. KYNA.  57.  Ratemeter record; spinal cord in vivo: Q U I S , L-GLU, KAIN and N M D A vs. M t r a n s - C P A .  64.  Ratemeter record; spinal cord in vitro: Q U I S , QUIN and N M D A vs. APV, A C R A and KYNA.  75.  Dose-response curves for K Y N A and APV against Q U I S , Q U I N , KAIN and NMDA.  78.  - x-  Figure 14.  Ratemeter records; spinal cord in vitro:  A. D-cis-ACPD, NMDA and L-cJs-ACPD vs. A P V a n d B. D-cjs-ACPD, N M D A and D-lmDS-ACPD vs. KYNA.  Figure 15.  Figure 16.  Figure 17.  Figure 18.  page  84.  Ratemeter records; spinal cord in vitro: A. Q U I S , 2,5-PyrDA and N M D A vs. A P V and B. Q U I S , 2,5-PyrDA and N M D A vs. KYNA.  89.  Ratemeter records; spinal cord in vitro: A. Q U I S , 2,6-PyrDA and N M D A vs. A P V and B. Q U I S , 2,6-PyrDA and N M D A vs. KYNA.  90.  Ratemeter records; spinal cord in vitro: Q U I S a n d N M D A vs. 2,4PyrDA and A P V  94.  Diagram of: Q U I S a n d N M D A receptor templates.  - xi -  133.  Acknowledgements T h a n k s are due to many for assistance, support and guidance during my studies: to Drs. Martin Peet and Ken Curry for their continuous support; to Dr. T o n y Pearson for chairing my committees and providing e n c o u r a g e m e n t ; to Mrs. Y v o n n e Heap for her excellent technical assistance; to Dr. John Church for his help during the writing of this thesis;  to Mr. Rheinhold W e b e r and Mr. Harry  Kohne for their collaborative creative efforts during the design, construction and maintenance of the tissue baths and to Mr. Rob Johnson for his efforts towards making the slice work. For their participation on my behalf appreciation is also due to the other m e m b e r s of my advisory, comprehensive a n d examination committees. Additional a c k n o w l e d g e m e n t belongs to the many other m e m b e r s of the Department of Physiology who made my time with t h e m both fruitful a n d enjoyable. Particular thanks are due to Drs. John Brown and David Godin for showing me the finer points of the recreational pursuits in which they individually excel. For their support and understanding special thanks goes to Julie and Trystan.  Finally, for his many efforts on my behalf and his outstanding example of excellence in science my appreciation is extended to Dr. Hugh M c L e n n a n .  - xii -  I. Introduction  A. History of excitatory amino acid research Historically, interest in amino acids in the nervous system w a s initiated by Hayashi (1954) who applied glutamate and aspartate topically to the cortex of dogs a n d observed overt convulsive behaviour.  In a review of his work (1959) Hayashi  d i s c u s s e d several possible roles for glutamate in the central nervous system in the light of results reported by Waelsch (1955) who found that glutamate w a s a c c u m u l a t e d in the brain, and a p p e a r e d to be taken up selectively across the blood brain barrier. Although Hayashi stated that convulsions produced by topically applied glutamate are physiological rather than pharmacological (since the threshold concentration w a s similar to the actual "wet weight" glutamate content of the brain, ie. it w a s applied at a physiological concentration), he a r g u e d mainly against the proposition that glutamate is a major excitatory transmitter because of the apparent involvement of glutamate in the energy supply of the brain. It was then thought that glutamate w a s an energy source for the central nervous system (secondary to glucose). However, on the basis that topically applied glutamate produced convulsions with a delay of 19 minutes, Hayashi did postulate that glutamate w a s linked metabolically to a c o m p o u n d which w a s the elusive excitatory transmitter. The discovery in 1959 by Curtis, Phillis and Watkins that y-aminobutyric acid (GABA; a neutral amino acid) could alter the electrical activity of neurones led to  1  the idea that the other amino acids, in particular aspartic, glutamic (Fig. 1, A & B) and cysteic, may in fact have roles other than metabolic ones in the central nervous s y s t e m . Systematic investigations into the excitation of central neurones by amino acids began in 1960 with the e n d o g e n o u s and readily available naturally occuring substances (Curtis et al., 1960a). That e x o g e n o u s and synthetic analogues of glutamate a n d aspartate could possess even greater abilities than glutamate itself to excite neurones w a s discovered early in the '60's, and by 1963 several basic structural requirements for excitation were recognized. T h e ability to excite "lay in the possession by a c o m p o u n d of one amino and two acidic groups, the latter being separated from each other by a distance equal to that occupied by a chain of two to three carbon atoms, whilst the amino group should optimally be situated in the alpha position with respect to one of the acidic groups" (Curtis and Watkins, 1963). In addition, Curtis and Watkins (1963) reported that the D-isomers of most of the c o m p o u n d s tested were of a similar potency to their L counterparts, but that the addition of a methyl group to the nitrogen of D-aspartate resulted in the extremely potent excitant N-methyl-D-aspartate (NMDA). With hind-sight, the basic structural requirements elucidated in the early 1960's are compatible with current models. Curtis and his colleagues were convinced, however, that the action of glutamate and other excitatory amino acids was completely non-specific and "that the site of action is a fundamental structural component of the m e m b r a n e s of all central neurones" (Curtis and Watkins, 1963). The criteria for identification of transmitters at that time were based entirely  2  0  O  ®  CO„H  ®  A  CCLH  ©  NH  N O  L-glutamate  L-aspartate  L-quisqualate  CO„H  ©  CCLH CH,  CCLH  H0 C  N ' H N-methyl-D-aspartate 2  ©  CO„H  NH.  HC 3  C0 H 2  a -(para-fluorophenyl)glutamate  a-methylglutamate  Figure 1.  Diagrammatic representation of the structures of A,. L-aspartate, EL. L-glutamate,  L-quisqualate, CL L-a-kainate,  E N-methyl-D-aspartate,  F. a-methylglutamate and  G^. a - p a r a - f l u o r o p h e n y l g l u t a m a t e  3  on what w a s k n o w n of acetylcholine; thus an excitatory transmitter should have specific actions only on those neurones which were e x p o s e d to it physiologically. That glutamate w a s able to excite virtually every neurone tested, even the cholinoceptive Renshaw cells of the spinal cord, posed a problem a n d s e e m e d to indicate that glutamate w a s unable to fulfill this criterion. Furthermore, Curtis, Phillis and Watkins (1960a) s h o w e d that the time-courses of amino acid excitations, in particular the "intervals preceding the cessation of cell reponses after the termination of the iontophoretic currents", (Curtis et al., 1960a) were identical for the D & L forms of glutamate, aspartate a n d cysteate. The time courses were not altered in the presence of a large variety of e n z y m e inhibitors. This argued against a transmitter role for amino acids since there were apparently no catabolic e n z y m e s , analogous to acetylcholinesterase, present to terminate the activity of the excitants. Furthermore, it was thought unlikely that any e n z y m e s present could a c c o m m o d a t e the variety of amino acid structures known to be active.  Curtis  (1962) also presented evidence showing that depolarization of motoneurones by glutamate had equilibrium potentials different from those of synaptically evoked depolarization. This w a s thought to be definitive evidence against any claim that glutamate w a s the excitatory transmitter for the monosynaptic reflex, since it failed to mimic the e n d o g e n o u s synaptic response. T h e opinion that glutamate does indeed act as an excitatory neurotransmitter in the brain a n d spinal cord w a s not widely held until the 1970's, a n d even then the debate continued. A key discovery which aided the change in opinion regarding  4  glutamate occurred in the late 1960's, but w a s largely overlooked at the time, was the finding that glutamate w a s compartmentalized in neurones, and existed as two pools linked only indirectly (Berl et al., 1961). A second important observation was made by Bennet et al. in 1973, who s h o w e d that many of the amino acids, including glutamate, aspartate and cysteate, were rapidly r e m o v e d from the extracellular fluid of the brain a n d spinal cord by neurones and glia.  It is now  believed that this high affinity uptake system acts to terminate the actions of the amino acid transmitters. More recent experiments involving the use of excitatory analogues of glutamate have shown t h e m to mimic accurately the postsynaptic m e m b r a n e events during synaptic transmission in the spinal cord a n d brain. In addition, it is now possible to block synaptic transmission in the C N S with potent amino acid antagonists at doses which do not affect depolarizations elicited by other agonists but which do attenuate the actions of an exogenously applied specific amino acid excitant. Finally, the release of glutamate from presynaptic terminals has been convincingly d e m o n s t r a t e d (see for example Potashner, 1978). It is now believed that the major criteria for acting as a transmitter are met by glutamate: it is present in, and released from presynaptic neurones under conditions of physiological stimulation (see for review F o n n u m , 1984); it acts post-synaptically in a manner similar to the endogenously released s u b s t a n c e , including displaying a comparable sensitivity to antagonists; and the response is rapidly terminated by a system for its removal from the a r e a of the  5  synapse. S o m e of this evidence will be discussed in more detail later in this section. T h e characterization of the receptor(s) for the amino acids did not proceed rapidly, a n d not until the late 1960's did any major d e v e l o p m e n t s occur.  McLennan  et al. (1968) and Duggan and Johnston (1970) then s h o w e d that regional differences existed in the relative potencies of the various glutamate analogues available, which started a debate regarding the possible existence of multiple receptors for the amino acids. In 1972, Haldeman a n d M c L e n n a n (1972) found support for the multiple receptor theory by showing L-glutamate diethylester (GDEE) to have different effects on the excitations elicited by various amino acids. Specifically, G D E E w a s found to block the excitatory action of L-glutamate (L-GLU) more effectively than that of DL-homocysteate in the thalamus a n d spinal cord. T h e n in 1978, M c L e n n a n and Hall reported that a long chain glutamate analogue, D-a-aminoadipate ( D a A A ) , had an antagonistic effect opposite to that of G D E E in that N M D A a n d DL-homocysteate were blocked more effectively than L-GLU and kainate (KAIN). This a n d subsequent studies in the thalamus a n d spinal cord of rats s h o w e d that w h e n the agonists were ranked according to susceptibility to blockade by G D E E and D a A A , they appeared to fall into two or possibly three groups thus providing substantial support for the multiple receptor theory ( M c L e n n a n a n d Hall, 1978). Several other long chain analogues, including D-aaminopimelic a n d D-cc-aminosuberic acid (Davies and Watkins, 1979), were also tested in the late 1970's and were found to be potent antagonists of amino acids  6  with actions similar to D a A A .  This prompted the obvious suggestion that the  extension of the carbon chain separating the co-carboxyl from the a-ionic groups b e y o n d the length of glutamate, a n d a D-configuration, were the structural characteristics necessary for antagonism of amino acids in the C N S . An exception to this t r e n d , 1-hydroxy-3-amino-2-pyrrolidone (HA-966), a cyclic analogue (5m e m b e r e d ring) devoid of carboxyl groups w a s first tested by Davis a n d Watkins in 1972, a n d found to be a moderately potent antagonist of amino acids. A subsequent study by Evans et al., 1978 s h o w e d that HA-966 acted in a similar fashion to D a A A (being more potent against NMDA-like activity than against that of other agonist) despite its lack of carboxyl groups a n d its cyclic structure. The theory that more than one "receptor" mediates the actions of the excitatory amino acids gained support, and efforts were increased towards finding specific antagonists for the proposed receptors. Prior to 1 9 8 1 , several antagonists were discovered and reported, but none had a greater impact on amino acid research than did 2-amino-5-phosphonovalerate, discovered by Davies et al., (1981b) and separated into its L (inactive) and potent D-isomers by Stone et al. (1981) and by Davies a n d Watkins (1982). This c o m p o u n d was first e x a m i n e d as a racemate in the spinal cord in vivo, and was found to block specifically excitations elicited by the iontophoretic application of N M D A while having little effect on firing c a u s e d by other excitant amino acids or acetylcholine (Davies et al., 1981). Also discovered in 1981 w a s the conformationally restricted antagonist cis-2.3-piperidine dicarboxylate (PDA; Fig. 2, F.) which was first e x a m i n e d by Davies et al. (1981a).  7  Figure 2. Diagrammatic representation of the structures of A.- P.: D- and L-, cis.- a n d t r a n s - 1 - a m i n o - 1 , 3 - c y c l o p e n t a n e dicarboxylate A , D-cis-ACPD; E L-trans-ACPD: C , D-transA C P D ; L I L-cis-ACPD; E (+) and H t r a n s - C P A a n d F. cis-2.3-piperidine dicarboxylate.  8  They reported it to be largely non-specific reducing NMDA-elicited firing of frog and cat spinal neurones only slightly more than those of quisqualate (QUIS) and KAIN. In addition they s h o w e d that when ejected onto motoneurones in the neonatal rat spinal cord in vitro. PDA c a u s e d depolarizations and firing that w a s A P V sensitive and therefore thought to be mediated by N M D A receptors. T h e s e studies followed the discovery by Evans et al. (1977) that N M D A - i n d u c e d excitation of frog and neonatal rat spinal neurones could also be selectively reduced by m a g n e s i u m ion ( M g ) , a finding which w a s subsequently demonstrated in the cat spinal cord in 2 +  vivo by Davies and Watkins (1977). By 1981 considerable evidence existed suggesting that at least two receptors were responsible for the actions of the amino acids in the spinal cord. In addition, a third receptor mediating KAIN excitations in the spinal cord w a s proposed, since these were relatively insensitive to M g , 2 +  D-(-)-2-amino-5-phosphono-valerate (APV) and G D E E , but w e r e blocked along with N M D A by the dipeptide y-D-glutamylglycine 1981).  (7DGG;  Davies and Watkins,  By inference t h e n , using A P V to block NMDA-like c o m p o u n d s , y D G G to  block N M D A - a n d KAIN-like c o m p o u n d s , and G D E E with a greater effect against L-GLU and other c o m p o u n d s including Q U I S , three receptors c o m m o n l y referred to as N M D A , KAIN and Q U I S receptors, are now believed to be responsible for the bulk of the excitatory actions exhibited by amino acids and amino acid-like c o m p o u n d s in the spinal cord and presumably elsewhere in the central nervous system (Fig. 1, C, D & E). Certain precautions are necessary, however, when interpreting results from different areas of the nervous system because of the  9  increasing evidence, which will be dealt with extensively in this thesis, for differences in the structural requirements for activation of the amino acid receptors in various regions. For example, M c L e n n a n and Liu (1981), reported that the excitation of spinal cord neurones by a racemic mixture of trans-1-amino-1,3cyclopentane-dicarboxylate (±trans-ACPD) was relatively insensitive to any of the antagonists y D G G , A P V and G D E E . Based on these results a fourth receptor for the a m i n o acids in the spinal cord w a s proposed. An additional line of research into the excitatory amino acids in the C N S was initiated by Anis et al. (1983), who e x a m i n e d the effects of a pair of dissociative anaesthetics, phencyclidine and ketamine, on excitatory amino acid responses of dorsal and ventral horn neurones. T h e s e c o m p o u n d s had been s h o w n previously to block L-GLU in the hippocampus, a m y g d a l a and thalamus (Sinclair and T i e n , 1979) a n d polysynaptic responses in the spinal cord (Tang a n d Schroeder, 1973). Anis a n d his colleagues (1983) s h o w e d that both of these c o m p o u n d s selectively reduced NMDA-elicited firing of spinal cord neurones at doses which had little effect on activity induced by QUIS or K A I N . Ketamine in particular has been used extensively in research since the majority of the antagonists which are analogues of glutamate will not cross the blood brain barrier w h e r e a s ketamine will.  B. Other e n d o g e n o u s excitatory amino acids and antagonists T h e recent addition of the tryptophan metabolites quinolinate (QUIN) and kynurenate ( K Y N A ) ; Perkins and Stone, 1982; Fig. 3, A and F) to the list of neurally  10  active amino acid analogues has s p a w n e d a v o l u m e of research into their possible roles as an e n d o g e n o u s amino acid agonist and antagonist respectively.  K Y N A is  a potent, conformationally restricted antagonist of c o m p o u n d s which act similarly to N M D A or K A I N , while QUIS-like c o m p o u n d s are less affected. For Q U I N , no uptake or enzymatic degradative system has been described, nor has release of Q U I N from nerve terminals been d e m o n s t r a t e d ; nevertheless a potential role as a modulator cannot be ruled out. Perkins and Stone (1982) suggested that neurally active m e m b e r s of the kynurenine metabolic pathway may potentially "play a part in the overall control of C N S excitability..."; in particular, that the balance between extracellular levels of Q U I N and KYNA may conceivably provide a long-term regulation of neuronal excitability. Acridinate (ACRA, Fig. 3, E.; Curry et al., 1986) a synthetic analogue closely related to K Y N A is also an antagonist of amino acid excitations. It is, however, completely non-specific reducing the responses to Q U I S , KAIN and N M D A comparably. A s early as 1960 (Curtis and Watkins, 1960b) the e n d o g e n o u s sulphur containing cysteic, cysteic sulphinic and homocysteic acids were known to be potent a m i n o acid excitants.  Interest in these c o m p o u n d s as possible e n d o g e n o u s ligands  for N M D A receptors has been recently rekindled due to the results obtained by Do et al. (1986) w h o s h o w e d cysteine sulfinic and homocysteic acids to be released in a calcium dependent manner from a variety of brain regions. Convincing evidence for their participation as C N S excitatory transmitters has not yet been a c c u m u l a t e d , but neither have results been reported to refute this suggestion.  11  ®  CO. H  N  2  2  C0 H 2  picolinate  phthalate  quinolinate  OH  C0 H  N  C0 H H0 C 2  N  C0 H 2  2,4-PyrDA  N kynurenate  2  ®  2  (G.  C0 H  N acridinate  2  3-hydroxypicolinate  Figure 3.  ©  C0 H  C0 H  N  ®  CO. H  ®  N  C0 H 2  H  0  c  2,6-PyrDA  2,5-PyrDA  Diagrammatic representation of the structures of & 2,3-pyridine dicarboxylate (quinolinate), E. 1,2-benzene dicarboxylate (phthalate), CX 2-pyridine carboxylate (picolinate), LI 3-hydroxy-2-pyridine carboxylate (3-hydroxy picolinate), E 2,3-quinoline dicarboxylate (acridinate), F, 4-hydroxy-2-quinoline carboxylate (kynurenate) and G^. 2,4-pyridine dicarboxylate (2,4-PyrDA), tL 2,5-pyridine dicarboxylate (2,5-PyrDA) and I 2,6-pyridine dicarboxylate (2,6-PyrDA).  12  C0 H 2  C. Intracellular studies in the hippocampus and cortex T h e d e v e l o p m e n t of the hippocampal and cortical slice preparations has permitted a more detailed characterization of the three "archetypal" (QUIS, KAIN a n d N M D A ) amino acid receptors by intracellular recording from neurones in these regions of the C N S . In pyramidal cells of the hippocampal CA1 region, the amino acid agonists can be characterized by the pattern of depolarization a n d firing which they elicit in addition to differential antagonism by A P V and KYNA. W h e n exposed to low iontophoretic doses of NMDA-like c o m p o u n d s , these neurones display a slowly rising depolarization followed by a bursting pattern of firing consisting of "tetrodotoxin (TTX)-sensitive spikes (usually three to four) s u p e r i m p o s e d on an underlying depolarizing shift in m e m b r a n e potential" ( Flatman et al., 1983; Peet et al., 1986), usually followed by a p r o n o u n c e d hyperpolarization. With higher iontophoretic d o s e s causing depolarizations greater than 15 mV, a transition occurs from the bursting pattern of firing to high frequency spikes superimposed on the depolarization.  Continued application of high levels of NMDA-like c o m p o u n d s  often causes extreme depolarization and spike inactivation (Peet et al., 1986). This /entire sequence of response is blocked by A P V and KYNA. W h e n e x p o s e d to KAIN-like c o m p o u n d s , the cells display TTX-sensitive spikes upon a slowly rising depolarization which increases throughout the application of the agonist and often leads to inactivation of the spike-generating mechanism. These effects are potently blocked by K Y N A but not by A P V , and several conformationally restricted c o m p o u n d s which evoke this type of firing are known (Curry et al., 1987).  13  Finally,  w h e n e x p o s e d to QUIS-like c o m p o u n d s , including L-GLU, the neurones display TTX-sensitive spikes and a depolarization which reaches a plateau quickly and does not increase further even when excess agonist is applied.  D. Excitatory amino acid receptor channels In vitro studies have also provided insight into the m e c h a n i s m s of activation by the three classes of amino acid excitants. Using whole-cell patch-clamp recording a n d voltage-clamp techniques, Mayer et al. (1987) s h o w e d that w h e n cultured spinal cord neurones loaded with the C a - s e n s i t i v e dye, arsenazo III, were 2+  c l a m p e d near resting levels of polarization, with u M concentrations of M g , N M D A 2 +  c a u s e d a large influx of C a in intracellular C a . 2 +  2 +  while Q U I S and KAIN elicited much smaller increases  The N M D A - i n d u c e d C a  2 +  influx b e c a m e voltage-sensitive  with physiological (mM) levels of M g , peaking at -30 mV. By clamping TTX2 +  treated neurones at various levels (-70 to +70 mV) in the absence of agonists, they found the voltage-sensitive C a  2 +  influx to peak at +10 mV. These findings, and the  fact that increased extracellular M g  2 +  reduced NMDA-like excitations without  affecting those elicited by agonists of the other two classes, suggested that the c h a n n e l o p e n e d by NMDA-like agonists permitted the flow of s o d i u m , potassium, calcium a n d m a g n e s i u m ions, while the channel(s) o p e n e d by Q U I S - a n d KAINlike c o m p o u n d s were more selective in that divalent cations were excluded.  The  studies mentioned previously, support suggestions that calcium ion influx may be involved with the well known toxic effects of this class of excitant, and that neuronal  14  death resulting from ischaemia may in part result from amino acid-induced toxicity. In addition, the involvement of excitatory amino acid-induced calcium ion influx in long-term potentiation, first proposed by Baudry and Lynch, (1980) underscores the importance of studies involving N M D A and N M D A antagonists (see for review Collingridge a n d Bliss, 1987). The ever-increasing use of cultured neurones a n d techniques such as patchc l a m p recording has a d d e d greatly to our knowledge of the m e c h a n i s m s underlying the excitation of neurones by amino acids. Recent studies by Jahr and Stevens (1987), and by Cull-Candy and Usowicz (1987a, b) have described the actions of low concentrations of excitatory amino acids on small patches of m e m b r a n e from cultured hippocampal and cerebellar neurones respectively. Using outside-out patches, Jahr and Stevens have found four distinct singlec h a n n e l currents e v o k e d by application of glutamate or its analogues; channel openings resulting in each of these currents appear to be dependent on which analogue is being applied. If these currents were the result of activation of four distinct receptor c o m p l e x e s , one would expect to find patches lacking one or more of the conductance states, a n d in patches where two or more of these complexes were present, one should expect to see currents resulting from the simultaneous opening of two or more of the channels.  Instead, these researchers have observed  direct transitions between each of the four conductance states in a single patch on several occasions, suggesting that all the various activities of the identified amino acid receptors may be accounted for by one complex molecular entity with three or  15  more separate binding sites. The largest conductance state, capable of permitting the passage of divalent cations, has a much higher rate of opening w h e n exposed to NMDA-like agonists, and spends proportionately more time in the open state w h e n bathed in magnesium-free m e d i u m . T h e s e characteristics are not observed for the lower conductance states seen more often with Q U I S - and KAIN-like agonists. Also using outside-out patches, but from cultured cerebellar neurones, Cull-Candy and Usowicz (1987a, b) have demonstrated a clear difference between the range of c o n d u c t a n c e states seen in these cells c o m p a r e d with hippocampal neurones. They have identified five different conductance states, a n d have observed transitions between all five, suggesting again that a complex consisting of one or two channels and two or more separate binding sites mediated the actions of the amino acids in these cells. Observed in both the hippocampal a n d cerebellar neurones, however, is the largest conductance state which results from N M D A activation, a n d which shows increased open times with magnesium-free medium.  E. Synaptic transmission in the spinal cord The potential roles of glutamate and aspartate as transmitters in the spinal c o r d were also being e x a m i n e d as early as 1972. Haldeman a n d M c L e n n a n (1972) d e m o n s t r a t e d that iontophoretically applied G D E E simultaneously blocked dorsal root stimulated monosynaptic excitations and L-GLU-induced firing of spinal  16  interneurones, while firing induced by L-aspartate (L-ASP) w a s largely unaffected. Biscoe et al. (1977), Hall et al. (1977) and Davies and Watkins (1978), reported that D-a-aminoadipate ( D a A A ) ejected at only slightly higher iontophoretic currents than those required to block NMDA-excitations (38 nA vs 26 nA), blocked the dorsal root stimulation of R e n s h a w cells in the spinal cord, while the cholinergic excitation induced by ventral root stimulation w a s unaffected. T h e results outlined a b o v e ( D a A A - & APV-sensitive and GDEE-sensitive receptors) were clarified in 1983 by Peet et al. w h o s h o w e d that both the polysynaptic excitation of dorsal horn neurones and the dorsal root excitation of Renshaw cells of cat spinal cord were blocked by a range of N M D A antagonists including DL-APV.  Neither were the  dorsal root-elicited monosynaptic excitations of dorsal horn neurones sensitive to antagonism by DL-APV nor were they blocked by PDA, a c o m p o u n d which attenuated KAIN-induced firing almost to the same extent as that of N M D A (Davies et al., 1981a). Studies by Davies and Watkins (1983, 1985) in the rat spinal cord in vivo also suggested that the low threshold primary afferent monosynaptic input (la) onto motoneurones w a s mediated by receptors insensitive to N M D A antagonists, and this conclusion is supported by the more recent intracellular studies by Jahr and Y o s h i o k a (1986) using hemisected neonatal rat spinal cord. Furthermore, in the cat, iontophoretic doses of A P V much higher than those needed to abolish completely the activity of iontophoretically applied N M D A did not significantly reduce the la excitatory postsynaptic potential (EPSP) recorded intracellular^ from motoneurones in vivo (Flatman et al., 1986). In the cat, therefore, it appears that  17  n o n - N M D A receptors may also be involved in the monosynaptic input onto motoneurones.  In s u m m a r y , monosynaptic inputs onto dorsal horn cells and  m o t o n e u r o n e s a p p e a r to be mediated by n o n N M D A - r e c e p t o r s while polysynaptic dorsal root inputs onto both dorsal horn cells and R e n s h a w cells probably involve N M D A receptors. Recent intracellular recordings by King et al. (1987) of dorsal horn cells in transverse slices of neonatal rat spinal cord have s h o w n that orthodromic stimulation of dorsal roots elicits (in 6 4 % of neurones tested) excitatory responses consisting of short- a n d long-lasting c o m p o n e n t s . The short c o m p o n e n t w a s an APV-insensitive fast-rising E P S P with typically one or two TTX-sensitive spikes s u p e r i m p o s e d , while an APV-sensitive longer latency "after depolarization" follows lasting up to 200 msec. It appears, therefore, that amino acid receptors play important roles in the excitatory transmission of sensory information in the dorsal horn a n d also in the mediation of motor reflexes in the ventral horn.  F. Synaptic roles in the hippocampus and cortex The hippocampus is a highly organized part of the higher C N S , the pathways of which have been well studied. It has two main inputs, the perforant pathways projecting to the dentate granule cells from the ipsilateral entorhinal cortex, and the Schaffer-collateral commissural pathways (SCC) projecting from the C A 3 to the CA1 pyramidal neurones of the ipsilateral and contralateral hippocampi.  An  additional internal pathway, the mossy fibres, project from the dentate granule cells  18  to the C A 3 pyramidal neurones. A study by Collingridge, Kehl and M c L e n n a n (1983a) described amino acid excitation of hippocampal neurones and synaptic transmission in the major pathways of the hippocampal slice, in vitro, using a range of excitant amino acid antagonists. They found that APV specifically and potently blocked N M D A elicited firing of hippocampal neurones, a n d that 7 D G G w a s capable, at slightly higher iontophoretic doses than required to block N M D A and KAIN-induced firing, of also consistently blocking QUIS a n d L-GLU elicited activity of any CA1 hippocampal neurone. Having thus established the criteria to distinguish between N M D A and n o n - N M D A receptor mediated firing, they p r o c e e d e d to examine the major synaptic pathways of the hippocampus a n d their susceptibility to these two antagonists. None of the excitatory inputs onto hippocampal pyramidal neurones could be blocked by DL-APV at "doses" which block N M D A , but 7 D G G w a s effective at reducing these inputs at doses which reduced Q U I S a n d KAIN excitations. A subsequent study (Collingridge et al., 1983b) concentrated mainly on the S C C input onto CA1 pyramidal neurones. They found that the long-term potentiation (LTP) of synaptic efficacy resulting from high-frequency stimulation (100 Hz for 1 sec) of this pathway w a s blocked by APV, despite the fact that A P V had little or no effect on the extracellularly recorded field E P S P . It appears then, that although N M D A receptors are not involved directly in low frequency transmission in the hippocampus, they are important during the induction of LTP. Wigstrom and Gustafsson (1984) discovered that the application of G A B A antagonists promoted LTP induction. Specifically, while recording  19  extracellularly from the dendritic region (stratum radiatum) of the CA1 area, and stimulating the S C C at various frequencies and durations, they found a current source in the dendritic region which was e n h a n c e d by picrotoxin (a G A B A antagonist) and d e p r e s s e d by APV. These findings along with those of Collingridge et al. (1983a, b) led Wigstrom a n d Gustafsson (1984) to suggest that tetanic stimulation of the excitatory pathways sufficiently depolarized the cells so as to remove the voltage-sensitive M g influxes of C a . T h e C a 2 +  2 +  2 +  blockade of N M D A channels leading to large  is thought to be responsible, at least in part, for the  induction of LTP. Intracellular recordings from pyramidal cells of the s o m a t o s e n s o r y cortex of the rat in vitro, have been used by T h o m s o n (1987) and demonstrate that excitatory inputs onto these cells are mediated in part by N M D A receptors. In coronal slices of cortex maintained under standard in vitro conditions, the response due to the iontophoretic application of N M D A and the synaptic response resulting from stimulation of the corpus callosum were similarly altered by changes in the M g  2 +  concentration and to the application of N M D A antagonists including A P V and ketamine. In contrast, O k a et al (1987) reported that thalamic excitation of extracellularly recorded cortical neurones in the cat in vivo was not attenuated by the iontophoretic application of APV at doses which blocked N M D A elicited activity. Instead, high doses of less selective antagonists such as K Y N A or PDA were required suggesting that this pathway was mediated by n o n - N M D A receptors. It appears t h e n , that one can tentatively assign callosal excitation of cortical  20  neurones to be mediated, at least in part, by N M D A receptors ( T h o m s o n , 1986) while the thalamocortical inputs may be mediated by n o n - N M D A receptors.  Recent  experiments by Avoli and Olivier (1987) using excised epileptic h u m a n neocortical slices maintained in vitro, suggest that N M D A receptors play an important role during seizure activity, since synaptically generated interictal spiking could be blocked by A P V at doses (50 to 100 uM) which block N M D A elicited activity, but did not reduce the intracellular^ recorded synaptic E P S P resulting from electrical stimulation of the underlying white matter. In summary, N M D A receptors appear to be involved in the induction of LTP in the hippocampus, a n d in certain of the excitatory inputs onto cortical neurones. N o n - N M D A receptors appear to be responsible for much of the low frequency transmission in the hippocampus and the thalamic inputs onto cortical neurones.  G. Actions of glutamate and aspartate As the list of potent amino acid agonists has grown the use of glutamate and aspartate to study the receptors which supposedly are responsible for their activities as transmitters has declined. Results obtained w h e n undertaking examinations of glutamate and aspartate are difficult to interpret due to several factors. First of all, both are rapidly taken up by neurones and glia (Logan and Snyder, 1 9 7 1 ; Bennet et al., 1973; Balcar et al., 1977; F o n n u m , 1984), while many analogues are not, and the efficiency of these uptake systems may not necessarily be similar from region to region in the C N S . Secondly, binding experiments have  21  s h o w n both glutamate and aspartate (the L-isomers) to be displaced by different g r o u p s of c o m p o u n d s acting specifically at more than one of the three archetypal amino acid receptors.  Electrophysiological evidence also suggests that the actions  of glutamate and aspartate are mediated via combinations of the different receptor t y p e s ; glutamate a n d aspartate are generally thought to be mixed agonists under most circumstances, although many exceptions to this generalization can be found (Davies et al., 1980). T h e roles played by L-GLU and L-ASP as transmitters, s o m e of which have already been m e n t i o n e d , are thought to occur via both N M D A and n o n - N M D A (presumably QUIS) receptors. There is little evidence for K A I N - r e c e p t o r mediated synaptic events in the m a m m a l i a n C N S , although Grillner et al. (1987) have s u g g e s t e d that in the lamprey cord certain of the pathways involved in locomotion are mediated by K A I N receptors. It is apparent that changes in extracellular M g  2 +  , kynurenate ( K Y N A ) or a  K Y N A - l i k e c o m p o u n d may influence the post-synaptic expression of a presynaptically released "mixed agonist" like glutamate (Perkins a n d Stone, 1982, 1984). C h a n g e s in the extracellular fluid content of excitotoxic amino acids have been considered as possible contributors to a variety of pathologies including epilepsy, Huntington's disease and ischaemic cytolysis (Coyle et al., 1981).  22  H. Quinolinate and other NMDA-like agonists Since the discovery of the tryptophan metabolite quinolinate (QUIN) in the C N S (Wolfensberger et al., 1983), it has b e c o m e increasingly evident that it should be considered a possible excitatory amino acid transmitter. Q U I N has been found, along with anabolic and catabolic e n z y m e s , in many C N S regions in a wide range of concentrations (Wolfensberger et al., 1983; Moroni et al., 1984; Foster et al., 1985) . Stone and Perkins (1981) s h o w e d that in the sensorimotor cortex of the rat in vivo. Q U I N had a potency about one quarter that of N M D A and one tenth that of Q U I S . This early study into the action of Q U I N also disclosed that in the cortex at least, it w a s indistinguishable from N M D A in its sensitivity to A P V . Subsequently, studies by Perkins and Stone (1983) and M c L e n n a n (1984) c o m p a r e d the potency of Q U I N in the cortex and spinal cord in vivo. In these studies, Q U I N w a s found to be at least five-fold less potent in the spinal cord than in the cortex, and few if any cells in the cord could be excited sufficiently to examine its pharmacological responsiveness. Therefore, there appears to be an anomaly: although N M D A is equipotent in cortex and spinal cord relative to Q U I S , ( Perkins and Stone, 1983; M c L e n n a n , 1984) and Q U I N appears to act at the N M D A receptor, Q U I N is extremely weak in the spinal cord. A more recent study examined the activity of Q U I N in the piriform cortex in vitro, and c o m p a r e d its action with L-ASP and N M D A (ffrench-Mullen et al., 1986) . A P V applied in the bathing medium at a concentration of 10" M blocked 6  NMDA-elicited spikes in preference to those c a u s e d by Q U I N and L-ASP.  23  These  authors c o m m e n t e d that the receptors responsible for the actions of Q U I N and N M D A in the piriform cortex have some c o m m o n characteristics but are pharmacologically distinguishable.  I. Cyclopentane and pyridine derivatives Identification of c o m p o u n d s reacting with N M D A receptors in the h i p p o c a m p u s has been accomplished primarily using the specific antagonist APV and observing the characteristic bursts produced, as described earlier.  An  extensive structure-activity analysis for these receptors in hippocampal CA1 pyramidal cells has recently been reported (Peet et al., 1987). Using a series of conformationally restricted and substituted analogues, it w a s s h o w n that only the 2,3 positioning of the carboxyl groups on a pyridine ring, as in Q U I N , permits activation of these hippocampal receptors. Furthermore the nitrogen atom present on N M D A (secondary amine) or on Q U I N (aromatic) w a s not essential for evocation of burst firing since phthalate (1,2-benzene dicarboxylate; Fig. 3, B) and itaconate (methylene succinate) were both w e a k agonists of N M D A receptors.  No  c o m p o u n d s have been reported to elicit NMDA-bursting which are not blocked by APV. T h e cyclopentane glutamate analogues were first e x a m i n e d as racemic mixtures (DL-cis- and DL-trans-1 -amino-1.3-cyclopentanedicarboxylate: ±cis- and + t r a n s - A C P D ) in the thalamus in vivo by McLennan and W h e a l (1978) and Hall et al. (1979) who found t h e m to be very potent agonists, cjs.-ACPD being more  24  sensitive to D a A A (the most potent N M D A antagonist at that time) and relatively insensitive to G D E E while t r a n s - A C P D not being greatly affected by either antagonist.  J. Structure of the dorsal horn T h e dorsal horn cells of the spinal cord are arranged in several layers (laminae I through VII; Rexed, 1952; see Fig. 4). They receive afferent information from the dorsal roots and relay it to other regions of the spinal cord, to the medulla and to the ventroposterolateral (VPL) nucleus of the thalamus. Processing of the afferent information within the dorsal horn is thought to occur through local circuits between cells of the substantia gelatinosa or SG (laminae II a n d III), which receive a proportion of the primary afferent fibre input, and the lower laminae (IV - VII). Although the histology and major pathways of the spinal cord are well k n o w n , and extensive somatotopic maps have been made for certain areas of the spinal cord, the local circuits a n d electrophysiological characteristics of the cells involved are largely unknown (Wall, 1967; Wilson et al., 1986). Information enters the spinal cord via the primary afferent fibres which have their cell bodies in the dorsal root ganglia. The peripheral processes originate from sensory receptors of all types, and centrally the fibres terminate in the dorsal horn of the cord. Within the cord, each central process divides into ascending and d e s c e n d i n g branches in either the zone of Lissauer (small myelinated and unmyelinated fibres) or the dorsal columns (large myelinated fibres).  25  These  branches give off collaterals w h e n they enter the cord and at various levels rostral a n d caudal to their point of entry. The largest and most medial of these fibres may a s c e n d in the dorsal columns as far as the medulla. Collaterals from these larger myelinated fibres pass through laminae I and II or bypass these laminae medially, a n d appear to terminate in the lower part of the SG on the dendrites of neurones with s o m a t a in lamina IV, or directly onto inhibitory interneurones of the lower laminae ( J a n k o w s k a and Roberts, 1972). The collaterals from the small myelinated a n d unmyelinated fibres for the most part terminate directly onto cells within lamina I a n d the SG (Cervero and Iggo, 1980). In general, the cells of the dorsal horn are arranged in a dorsoventral fashion. Their dendrites extend dorsally into the adjacent laminae, and their axons either travel ventrally to lower laminae, or pass into the white matter to a s c e n d or d e s c e n d to other levels of the cord, or directly to higher centres. T h e exceptions to this are the cells of lamina I (the marginal zone), which are spindle s h a p e d and lie parallel to the dorsal surface of the cord, with processes extending mediolaterally for several tens of microns (Cervero and Iggo, 1980).  K. In vitro slice preparations Since the initial studies by Li and Mcllwain in 1957, the in vitro slice preparation has been modified to suit a variety of applications in many areas of the nervous system. However, because of the relative softness of the tissue even at low temperatures, the adult mammalian spinal cord has proved difficult to prepare  26  for in vitro studies. T h e s e difficulties are especially evident w h e n transverse slices are attempted although Zieglgansberger and Sutor (1983) provided s o m e evidence for the feasibility of this preparation. Other laboratories are currently studying the spinal cord prepared from neonatal rats in vitro. Hemisected cord (Jahr a n d Yoshioka, 1985; Fulton a n d Walton, 1986), or horizontal (Murase et al., 1982) a n d transverse slices (Ma and D u n , 1985; Miletic a n d Randic, 1982; T a k a h a s h i , 1978) of the lumbar region have been used for both intra- a n d extracellular electrophysiological studies with the majority of the w o r k to date concentrating on the substantia gelatinosa, an area easily recognizable as a translucent band in slices less than 600 u M thick.  The use of neonatal tissue for  the study of the excitatory amino acid receptors in the spinal cord may not be entirely appropriate however, since a number of recent studies have s h o w n that several populations of spinal receptors such as those for 5-HT (Lau et al., 1985), G A B A (Saito et al., 1982) and glycine (Benavides et al., 1981) do not reach adult levels until the 14th, 15th and 30th postnatal day respectively. W e have therefore performed studies on spinal cord slices prepared from weanling rats (24 to 30 days old) rather than from neonatal animals. Several reviews available on slice preparations stress that the slice of nervous tissue should contain, intact, as many of the known synaptic pathways as possible (see for example Richards, 1981). Although many of the synaptic circuits in the dorsal horn are local, the ideal slice w o u l d have both the dorsoventrally oriented cells of the SG and lamina IV and V, and the rostrocaudally oriented  27  primary afferent branches in the zone of Lissauer and dorsal c o l u m n s , intact. This has led to the use of a dorsoventral longitudinal slice (sagittal) which extends rostrocaudally for,8 or more m m ; it should therefore contain at least three complete spinal s e g m e n t s of the lumbar enlargement of the rat cord. The advantages of this preparation lie in the potential for application of antagonists at a k n o w n concentration in the perfusion medium and in the opportunity for future intracellular investigation of spinal neurones. This preparation should also permit the study of synaptic activity both intra- and i n t e r s e g m e n t a l ^ within the spinal c o r d since several complete s e g m e n t s are contained within one slice.  II. Aims of the Present Studies  Excitatory amino acid receptors are involved in synaptic transmission in many regions of the m a m m a l i a n C N S . Several of the pathways mediated by these receptors have already been discussed, their functional significance ranging from the possible involvement of N M D A receptors in learning and memory (Collingridge, 1987) to the transmission of sensory and reflex information in the dorsal horn of the spinal cord (Headley et al., 1987). The primary aim of these studies w a s to contribute to the knowledge of how the amino acid receptors in the spinal cord differ, in terms of their structural requirements for activation, from those elsewhere in the C N S , a n d to provide a foundation for future study aimed at designing specific  28  antagonists for Q U I S , KAIN and Q U I N spinal cord receptors. In order to determine the structural requirements for activation of amino acid receptors in the spinal cord, in vivo a n d in vitro experiments were used to examine the activities of several groups of glutamate, aspartate and cyclic amino acid analogues. The rationale for synthesizing and testing the first group of c o m p o u n d s was based on the premise that substituents of a bulky aliphatic or aromatic nature may produce analogues of high potency either as agonists or antagonists.  Such  c o m p o u n d s may bind to the receptor a n d , because of the substitutions, be unable to activate, or conversely, may activate but may have difficulty disengaging from the receptor.  Several a-substituted analogues of glutamate were investigated including  the methyl, phenyl, a n d para-substituted phenyl variations (examples Fig. 1, F and G: page 3). Since the excitatory amino acids are believed to interact with their receptors in the spinal cord by a three-point attachment (the two acidic groups a n d the amino group), the relative position in space of these groups b e c o m e s exceedingly important to an understanding of the structural requirements of the receptors. A valuable c o m p o u n d to study w o u l d therefore be a potent agonist or antagonist which p o s s e s s e d a rigid structure with little possibility for conformational change.  The  cyclopentane-derived analogues of glutamate and aspartate are ideal for this type of investigation, a n d several different c o m p o u n d s of this type were e x a m i n e d (Fig. 2, A - E: page 8). A s with the a-substituted c o m p o u n d s , they were tested initially using the in vivo preparation. Because all of the isomers available had interesting  29  Full N a m e s and Abbreviations of All C o m p o u n d s T e s t e d  concentration (mM) abbreviation  full name of compound  -in Vivo  Agonists:  QUIS KAIN NMDA QUIN (DL)t-ACPD D-cjs-ACPD L-cjs.-ACPD L-trans-ACPD D-trans-ACPD L-GLU D-GLU L-ASP 2,5-PyrDA 2,6-PyrDA 3-HPA ITCA PHTA  *  -in vitro  *  source  quisqualate kainate N-methyl-D-aspartate 2,3-pyridine dicarboxylate (quinolinate) (DL)trans-l -amino-1,3-cyclopentane dicarboxylate D-cis-1-amino-1,3-cyclopentane dicarboxylate L-cis-1-amino-1,3-cyclopentane dicarboxylate L-trans-1 -amino-1,3-cvcloDentane dicarboxylate D-trans-1 -amino-1.3-cvclopentane dicarboxvlate L-glutamate D-glutamate L-aspartate 2,5-pyridine dicarboxylate 2,6-pyridine dicarboxylate (dipicolinate) 3-hydroxypyridine-2-carboxylate methylene succinate (itaconate) 1,2-benzene dicarboxylate (phthalate)  5 50* 50* 200 200 n.t. n.t. n.t. n.t. 500 500 500 n.t. n.t. n.t. n.t. n.t.  5 5* 20* 20 n.t. 200 200 200 200 100| 200 n.t. 200 200 200 200 200  Sigma Sigma Sigma Aldrich K. C. K. C. K. C. K. C. K. C. Sigma Sigma Sigma Aldrich Aldrich Aldrich Aldrich Aldrich  4-hydroxyquinoline-2-carboxylate (kynurenate) D-(-)-2-amino-5-phosphonovalerate 2,3-quinoline dicarboxylate (acridinate) 2,4-pyridine dicarboxylate (±)cjs_-2,3-piperidine dicarboxylate glutamate diethylester a-phenylglutamate a-methylglutamate a-(para-fluorophenyl)glutamate a-(para-chlorophenyl)glutamate (±)trans-1 -amino-1.2-cycloDentane dicarboxvlate Mtrans-1 -amino-1.2-cyclopentane dicarboxylate (+)ltaaa-1-amino-1,2-cyclopentane dicarboxylate  100f 10* 200 n.t. 100f 200 200 200 200 200 200 200 200  20* 2.5* 200 200 n.t. n.t. n.t. n.t. n.t. n.t. n.t. n.t. n.t.  Aldrich T. N. K. C Aldrich K. C Sigma K. C. K. C. K. C. K. C. K. C. K. C. K. C.  -not tested -synthesized by Dr. K. Curry -supplied commercially by Tocris Neuramin  * f  Antagonists:  KYNA APV ACRA 2,4-PyrDA PDA GDEE aPG aMG apFPG apCPG (±)tran$.-CPA (-)trans-CPA  (-t-)trans-CPA n.t. K. C T.N.  -in150mMNaCI -in75mMNaCI  Table 1. Complete list of all compounds tested; the abbreviations used in the text, their full name, concentrations used for iontophoresis in vivo and in vitro, and the source of the compound. All compounds dissolved in distilled water unless stated otherwise.  30  activities in vivo, the four isomers of cyclopentane glutamate (D- and L-, cjs- and trans-1 -amino-cvclopentane-1.3-dicarboxylate; D- a n d L-, cjs.- and f r a n s - A C P D ) were e x a m i n e d extensively in vitro. The two isomers of trans-cyclopentane aspartate, [(+) a n d (-)trans-1-amino-1,2-cyclopentane-dicarboxylate; (+) and (-)trans-CPA] were not synthesized in sufficient quantities to examine t h e m in vitro. T h e separated optical isomers of d s - 1 - a m i n o - 1 , 2 cyclopentane-dicarboxylate have proved difficult to synthesize and were not available to study. T h e third series of analogues e x a m i n e d were based on the a f o r e m e n t i o n e d e n d o g e n o u s excitant 2, 3-pyridine dicarboxylic acid (quinolinic acid; Q U I N , Fig. 3, A: page 12).  A lengthy study of the agonist activity of Q U I N in the spinal cord w a s done  in order to characterize its action with respect to the well-known agonists and antagonists. Additional experiments looked at several other pyridine derivatives including the 2,4-, 2,5-, 2,6- (Fig. 3, G-l: page 12) a n d 3,4-dicarboxylate variations. The substituted pyridines 3- (Fig. 3, D: page 12) and 4-hydroxy-2-pyridine dicarboxylate a n d 1,2-benzenedicarboxylate (Fig. 3, B) were also e x a m i n e d . A complete list of all the c o m p o u n d s used in these studies is shown in Table 1; the abbreviations used and suppliers of the c o m p o u n d s are listed for reference.  31  III. Methods  A. Spinal cord a n d cortex in vivo In vivo studies were carried out using male Wistar rats (200-350 g) anaesthetized with urethane (1.5 g - k g  -1  i.p.). A mid-dorsal incision of the skin from  the scapulae to the sacrum initiated the surgery. Parallel incisions of the muscle 2 to 3 m m on either side of the vertebral dorsal spinous processes were then made from the fat pad of the back ( T or T ) to the sacrum. T h e medial strip of muscle, along 9  1 0  with the spinous processes, w a s removed with scissors and toothed forceps.  Small  blunt scissors were then used to clear adhering muscle from the lateral edges of the vertebrae, and the dorsal surfaces were scraped clean using a periosteal elevator. Small Friedman rongeurs were then employed to make a w i n d o w in the rostral dorsolateral corners of the T10 vertebra permitting the insertion of small bone cutters to sever the vertebral laminae. The dorsal portion of the vertebra w a s removed exposing the underlying d u r a and spinal cord. This process w a s repeated, save the use of the rongeurs, for the remaining vertebrae. Exposed tissues, muscle and d u r a were periodically moistened with w a r m e d , o x y g e n a t e d artificial cerebrospinal fluid (ACSF) throughout the procedure. Under a dissecting microscope, the d u r a w a s r e m o v e d and/or reflected using fine forceps and iridectomy scissors. Large vertebral clamps were then placed on the most rostral and caudal e x p o s e d vertebrae to immobilize the c o l u m n a n d cord. The incised skin flaps were raised and tied to an oval frame in order to form a pool which was subsequently filled with w a r m paraffin  32  oil to prevent desiccation of the exposed tissues. A rectal probe a n d heating bed were used to maintain a body temperature of 35° C. Surgery to expose the cortex began with a rostrocaudal skin incision of 2.5 cm in length, 0.5 c m to the left of midline, exposing the cranium overlying the left sensorimotor cortex. Connective tissue a n d muscle were r e m o v e d , a n d the cranium w a s roughly w i p e d with a dry sponge a n d bone wax. A w i n d o w approximately 1.0 x 1.0 c m w a s made in the cranium using a dental drill. Care w a s t a k e n not to touch the underlying d u r a and cortex as slight disturbances often resulted in severe swelling a n d bleeding. As with the spinal cord, the d u r a was reflected, the skin flaps tied to a circular frame (about 4 c m in diameter) and w a r m paraffin oil w a s a d d e d to cover the e x p o s e d tissues.  B. Spinal cord slice preparation Male Wistar rats (50 - 100 g) were anaesthetized with urethane (1.5 g-kg- i.p.). 1  The spinal cord w a s e x p o s e d from T  1 0  to S i by dorsal laminectomy.  Using a small  pair of curved scissors, parallel incisions (2 to 2.5 mm) were made in the lateral portions of the most rostral e x p o s e d vertebra. The portion of vertebra between these two incisions w a s then carefully removed with small curved haemostats. The lateral aspects of this vertebra were removed with a small pair of Friedman rongeurs.  The  dorsal lamina of each subsequent vertebra w a s then removed using small bone cutters a n d curved haemostats as described for the in vivo preparation.  Bleeding  w a s carefully controlled with slivers of sponge or g e l f o a m , and all bleeding was  33  staunched before proceeding further into the preparation of the slices. The d u r a was reflected a n d the dorsal and ventral roots cut as close to their spinal origins as possible. Although care w a s t a k e n to avoid pressure upon the dorsal surface of the cord during surgery, the viability of the preparation w a s not c o m p r o m i s e d by the occasional light touch. The cord w a s then cooled by the gentle application of oxygenated A C S F at 3°C (3 - 4 ml). W h e n the anterior a n d posterior ends of the e x p o s e d cord were sectioned, the entire length could be transferred to a dish of A C S F at 3°C. After 60 - 90 s., during which time any adhering dura, blood clot or roots were carefully dissected away, the length of cord w a s placed on the chilled cutting stage of a Mcllwain tissue chopper. The stage w a s modified by the addition of a raised wall ca. 3 x 5 c m and 0.75 c m deep which permitted the tissue slices, once cut, to be s u b m e r g e d in A C S F prior to their removal to the recording c h a m b e r (see below). Using the blade of the chopper (a standard platinum-chrome d o u b l e - e d g e d razor blade), the ends of the cord were cut away leaving a 4 - 8 mm segment which included the bulk of the lumbar enlargement. This piece of cord w a s then realigned longitudinally, ventral side up. Eight or nine 400 urn slices were cut in quick succession, leaving each slice in place to provide support for subsequent slices. After cutting, the well was filled with A C S F (3°C), a n d the slices were gently separated with a fine paint brush. Each slice w a s transferred by wide mouth pipette onto the taut nylon mesh support of a low-volume, continuously perfused tissue chamber. The slices were maintained at the interface between a w a r m moist  34  atmosphere of 9 5 % 0  2  - 5% C 0  2  and A C S F at 33°C. Up to 4 slices per cord,  originating 600 - 1400 urn on either side of midline, d e m o n s t r a t e d a sharply-defined dorsal root region and substantia gelatinosa, as well as a consistently high degree of neural activity. The most medial and lateral slices contained very little grey matter a n d were discarded (Fig. 4). An incubation period of at least 90 minutes was necessary prior to the start of any experiment since extracellular recording from slices s h o w e d greatly reduced neural activity until this time. In general, a gradual increase in activity w a s observed for 3 h after preparation of the slices. No differences in viability or neuronal responses were observed between slices prepared from rats weighing less than 100 g; h o w e v e r d e c r e a s e d activity w a s observed in slices prepared from rats 125 g or larger. During the development of this procedure, a variety of different approaches to the problem of getting viable slices from the spinal cord of y o u n g adult rats were utilized. The initial attempts focussed on transverse slices of the lumbar or cervical enlargements or of the thoracic cord. Some success w a s experienced with all three of these approaches. Problems were encountered, however, with the longevity of the slice, a n d with the consistency of the preparation (ie. some slices would have few or no live cells, with no plausible explanation). W h e n e x a m i n e d using a high-power (500 X) dissecting microscope, transverse slices of cord in the tissue bath appeared  35  400n  Figure 4. A. Diagrammatic representation of the lumbar enlargement of the rat in cross section, and the approximate lines of passage of the chopping blade through the cord. B. Diagrammatic representation of one slice as it would appear during an experiment with the visible structures indicated.  36  to leak the cytoplasm of d a m a g e d cells or cut axons into the A C S F . In a slice 400 urn thick, it s e e m s probable that the cut ends of rostro-caudally oriented axons do not seal but rather empty themselves of cytoplasm, which must flow through and over the rest of the slice. This suggestion is supported by the observation that the viability of the dorsal horn cells d e p e n d e d on their relative orientation in the bath with respect to the flow of A C S F . On several occasions, simultaneously cut slices were placed in the bath, s o m e with the dorsal half upstream (ventral horns oriented t o w a r d s the suction) and others with the dorsal half d o w n s t r e a m (dorsal horns oriented t o w a r d s the suction). It w a s observed that the slices with their dorsal horns placed d o w n s t r e a m , such that the A C S F first flowed over the ventral horns, were routinely of poorer quality relative to the others. Longitudinal slices, on the other hand, w o u l d have many of the rostro-caudal axons intact for distances up to the entire length of the slice (6 - 8 mm). Microscopic examination of these slices once in the bath s h o w e d that s o m e leaking of cytoplasm still occurred, but to a lesser degree and for a much shorter time. Several successful experiments were done with slices cut, either longitudinally or transversely, from the cervical enlargement. The most successful approach to this preparation w a s to decapitate and surgically isolate the cord by laminectomy, keeping the cord chilled from the moment of exposure: The viability of the slices was inconsistent, however, for several possible reasons. The cervical enlargement is larger t h a n , and contains relatively more white matter than does the lumbar enlargement.  It w a s therefore more difficult to cut, and if the procedure did not run  37  entirely smoothly such that the time taken between decapitation a n d the slices arriving in the bath w a s much over 6 - 8 minutes, the results were unsatisfactory (these strict requirements were by comparison quite relaxed w h e n working with the lumbar enlargement). Viability w a s achieved more consistently with cervical slices from animals of 25 to 50 g. However, for the reasons given earlier the use of such y o u n g animals w a s unacceptable, and experiments with the cervical region were not performed. Slices of the thoracic cord of rats above 50 g. in weight were also successfully prepared. The thoracic cord, 2.2 - 2.6 m m in diameter by comparison with the lumbar enlargement (3.3 - 3.9 mm), w a s troublesome to work with. It w a s very difficult to get more than a single longitudinal slice with the correct orientation from each piece of cord. Transverse slices of thoracic cord were also prepared with s o m e success, although they still suffered from the problems outlined above. In addition, the small size of the dorsal horn and lack of cell numbers made it less than optimal for the studies undertaken here.  C. Maintaining spinal cord slices During the development of the slice technique, it b e c a m e apparent that the environmental requirements (ie. A C S F flow and 0  2  content, temperature and  humidity) were different and more rigid for the spinal cord than for the hippocampus. The tissue bath used for hippocampal slices in the laboratory was of a simple, single c h a m b e r design where the slice c h a m b e r a n d the c h a m b e r  38  holding the heated distilled water used to humidfy the 0 / C 0 2  2  were continuous  (Fig. 5). W h e n placed in the single c h a m b e r bath, slices of the spinal cord survived for 3 to 4 hours at most. In an effort to improve the viability of the slices, a slice c h a m b e r of completely new design w a s e m p l o y e d .  D. Design of tissue c h a m b e r T h e viability of spinal cord slices once introduced into the in vitro c h a m b e r d e p e n d s on an a d e q u a t e flow of w a r m e d a n d o x y g e n a t e d A C S F below, and moisture-laden 9 5 % 0  2  - 5% C 0  2  above. The amount of moisture carried by the  g a s e s (humidity) is dependent on the temperature of the fluid through which they are bubbled a n d is a critical c o m p o n e n t of slice maintenance which cannot be controlled in single c h a m b e r baths of conventional design. If the distilled water used to humidify the gases is separated from the tissue c h a m b e r it can then be maintained at a temperature different from that of the slices. Thus a two c h a m b e r bath w a s d e s i g n e d which permits saturation of the gases at any temperature prior to introduction into the slice chamber. The amount of condensation in the tissue c h a m b e r is then very accurately controlled by varying the temperature of the water used to humidify the gases. T h e construction of the bath itself is also unique. A heated base and outer sleeve of aluminum encases a tissue c h a m b e r constructed of lexan (Fig. 6). A pair of transistors, e m b e d d e d in the aluminum base, are used as a source of heat, the current to which is externally controlled by a microthermistor also e m b e d d e d in the  39  B.  ~—I A.  L  9.  a. 3:  A  A  A  A'V  A A A  A  A  A  A  A  A  A  A  A  A  6.  A  10.  -ACSF  -plexiglass  0  -distilled water  -ACSF tubing (left) -heating coil (right) TOP VIEW  scale  1.  -lexan ring and taut nylon mesh to support slices  2.  -airstone inlet for 95% 02 - 5% C02  3. -distilled water or ACSF 5.  4. -ACSF tubing  -nichrome wire heating coils (in teflon tubing)  6. -plexiglass bottom and sides  7. -plexiglass lid  8. -bubble stop  9. -suction chamber  10.-light source  Figure 5. Diagram (not to scale) of a single c h a m b e r tissue bath of conventional design. The c h a m b e r holding the water through which the gases are bubbled (A) a n d the tissue c h a m b e r itself (B) are contiguous.  40  H Q  -  A C S F  -silver  [s^j -aluminum  rj  -lexan  ES9 -transistor  H  -thermistor  TOP VIEW  not to scale  1.  -lexan ring and taut nylon mesh to support slices  2.  -oxygen chamber  3.  -inlet port for warm/moist 95% 02 - 5% C02  4.  -inner lexan sleeve  5.  -outer lexan sleeve  6.  -silver ground  8.  -tubing and inlet port for ACSF  9.  -aluminum baseplate and housing  10. -suction chamber  7.  -temperature probe and transistors  11. -light source  Figure 6. Diagram (not to scale) of a t w o - c h a m b e r e d tissue bath of novel design. The water through which the gases are bubbled is held in a separate container which is maintained at a temperature which provides the optimal humidity in the slice chamber.  41  base. T h e tissue c h a m b e r maintains an A C S F depth beneath the slices of less than 1.2 m m and has a total volume of less than 1 ml. The working a r e a is circular with a diameter of 18 m m permitting up to three electrodes to be introduced into the c h a m b e r at one time. T h e total volume of the tissue c h a m b e r from reservoir to suction, including the tubing is less than 3 ml. This permitted the application of c o m p o u n d s in the perfusate with rapid turnover of the fluid in the chamber. Tryptamine, which has a specific ultraviolet absorbance at 640 nm, w a s added to the perfusate in order to observe the rate of change of concentration of c o m p o u n d s applied. S a m p l e s of 6 ul. were taken every 30 seconds from the fluid surface at the centre of the bath, diluted to 1 ml., and the absorbance measured. Figure 7 shows that the concentration reached 66 % of the theoretical m a x i m u m within 3.5 minutes. W h e n applied in 10 ml. volumes at the standard rate of 1.5 ml.-min.- , the "washout 1  curve" shows that a maximum concentration of ca. 92 % w a s obtained, while a level of 66 % w a s maintained for approximately 6 minutes (Fig. 7). During some experiments consistent firing rates for two or three agonist cycles (see below) were not obtained with a 6 minute application (particularily w h e n 4 or more agonists were being tested at one time), and antagonists were therefore applied in volumes of 20 ml. or more. In these situations it w a s a s s u m e d that the m a x i m u m theoretical concentration of antagonist (100 %) w a s obtained and accounted for the observed reduction in the size of the responses.  42  100  T  90 • 80 • 70  •  60 • 50 • 40 • 30 • 20 • 10 • 0  • 0.5  1ll-T-r-T-rn, 2  3.5  5  6.5  8  9.5  11  12.5  14  time (minutes)  Figure 7. Bar graph of the absorbance (% of control) at 640 nm of tryptamine solution sampled (6 ul. every 30 sec.) from the middle of the perfusate surface. Stock solution of tryptamine w a s diluted in 10 ml. of A C S F which w a s applied at the standard rate of 1.5 ml.- min.- . Arrow indicates w h e n the 10 ml. volume of antagonist solution ends a n d is replaced by normal A C S F at the fluid pump. 1  43  E.  Microiontophoresis Seven barrelled glass microelectrodes prepared from o m e g a - d o t tubing were  p u r c h a s e d as blanks from Vancouver Scientific Glass Blowing. T h e blanks w e r e pulled on a vertical electrode puller, a n d broken back to a final diameter of 5 - 8 urn under visual control with a Leitz micromanipulator.  C o m p o u n d s were applied  iontophoretically with currents ranging from 1 to 100 nA from the outer 6 barrels of the 7-barrelled microelectrodes.  Microiontophoresis is based on the principle that  ions will move in solution w h e n a charge is applied to that solution, resulting in current flow. If the ion to be ejected is negatively charged at the pH used, and is the "only" ion in the solution, it can be a s s u m e d that any current resulting f r o m an applied charge is predominantly carried by the desired ion. T h e outer barrels containing the amino acids to be ejected had, in these experiments, resistances between 50 and 200 M f t . To prevent the barrels from having much higher resistances when attempting to apply very small amounts of a potent agonist or antagonist, the ion to be ejected comprised only a percentage of the total ionic strength of the solution. Thus for example, Q U I S w a s c o m m o n l y used at 5 m M in 150 m M NaCI. The total ionic concentration of the solution is therefore 155 m M , with the ion of interest comprising only about 3.2 % of the total (Table 1: page 30). Therefore, when ejecting QUIS with a current of 100 nA, the current carried by quisqualate molecules is approximately 3 nA.  Another  complication arises w h e n determining how much amino acid is leaving the iontophoretic barrel. Proportionately more of the current will be carried by the  44  chloride ions than by the amino acid due to the lower mobility of the latter.  Variability  in the iontophoretic efficiency of individual electrode barrels and other factors make it very difficult to use the technique of iontophoresis for quantitative estimates of the relative potencies of agonists and antagonists. Over a large number of experiments, however, using many electrodes a n d testing n u m e r o u s cells, it is possible to estimate the relative potency of an agonist based on the average firing rate achieved per nA of current applied, taking into account the concentration of agonist and NaCI in the solutions used. Antagonist potency can be more accurately a s s e s s e d by application in the superfusate, as will be explained later. Although current balancing w a s not u s e d , cells which displayed rapid changes in firing with the onset or offset of current were not included. A complete list of all c o m p o u n d s tested, and the concentrations used for iontophoresis both in vivo and  in vitro, is shown in Table 1  on page 30. T h e amino acid solutions for iontophoresis were adjusted to pH 7.6 8.0 using 0.1 M N a O H , allowing the c o m p o u n d s to be ejected as anions.  F. Recording electrical activity T h e extracellular electrical activity of dorsal horn and cortical neurones was recorded from the 4 M NaCI-containing central barrels of the electrode assemblies, a n d had resistances of less than 4 MQ. The electrical activity of the neurones was passed through a 100 gain preamplifier to an oscilloscope and w a s also displayed on a second oscilloscope after passing through a variable filter (Fig. 8). Action  45  raw signal filtered signal  r-  finaldata experimental parameters  Figure 8. A) Seven-barrel glass microelectrode. B) Piezoelectric microdrive with depth indicator and remote control. C) Iontophoretic current generator with a u t o m a t e d timer. D) 100 gain preamplifier g r o u n d e d to the perfusate of the tissue chamber. E) Channel A of oscilloscope # 1 . F) Variable filter. G) Oscilloscope #2. H) Winston Instruments rate meter / w i n d o w discriminator. E) Channel B of oscilloscope # 1 . I) Hewlett Packard thermal printer. J) Spikes - s e c . plotted on a chart recorder. Recordings were made of the electrical activity of neurones maintained in vitro at 33° C in a two-chambered tissue bath K), or in vivo with the animals body temperature maintained at 35° C by L) a rectal thermistor and heating bed. -1  46  potentials of 1 mV in height were routinely recorded both in vivo a n d in vitro: spikes from single neurones were isolated with a window discriminator and rate meter records were made. The numbers used to express the activity of the cells were counts of the action potentials generated during each agonist ejection period only.  No  estimates were attempted of the various times of onset or offset of cell activity. Care w a s taken however, to adjust the position of the electrode relative to the cell and magnitude of the ejection currents u s e d , to achieve rate meter peak heights and durations, as well as action potential numbers, that were similar for each agonist tested. This w a s not difficult to accomplish for most cells and for most agonists. Agonists were usually ejected for 10 or 15 seconds followed by a 10 or 15 second recovery period. A typical cycle would test two, three or sometimes four agonists against one or two antagonists. The agonists were applied in timed a u t o m a t e d s e q u e n c e s , while the ejection currents for the antagonists were turned on and off manually.  In general, three complete agonist cycles showing a minimum of variation  were c o m p l e t e d as controls prior to the addition of an antagonist. T h e antagonist w o u l d then be applied until the responses to one or more of the agonists w a s reduced by more than 5 0 % , or until two or more cycles showing little variation were complete. The antagonists were also applied in the superfusate in 10 ml. v o l u m e s at 1.5 m l - m i n - , which provided a constant concentration of the antagonist for about 6 1  minutes. O n occasion, recordings were made during which 4 or 5 agonists were cycled a n d topically applied antagonists only were u s e d : this necessitated using larger v o l u m e s of antagonist (as mentioned previously) to a c c o m m o d a t e the prolonged agonist cycle times.  47  IV. Results  A. Presentation of the d a t a Figure 9, A. shows a ratemeter recording of the activity of a single dorsal horn neurone in vivo elicited by ejection of Q U I S at 35 nA, L-GLU at 60 nA and N M D A at 40 nA. Each of these agonists were automatically applied in turn (indicated by horizontal bars beneath each peak) with 20 seconds of recovery allowed between each ejection period. The instantaneous firing rate of the cell c a n be estimated by comparing the ratemeter peak with the vertical scale bar on the left. T h e antagonist, PDA in this case, w a s turned on manually and applied for 4 complete agonist cycles; the horizontal bar above indicates the final cycle (three ratemeter peaks) recorded during the ejection of the antagonist. Only the spikes elicited during the ejection of the agonists were counted a n d used to represent the activity of the cells. The ratemeter record, however, shows all cell activity before, during a n d after the application of each agonist. The tabular d a t a is presented in three forms. First of all, it is depicted as the m e a n % reduction of control firing, pooled for all cells (Table 2 middle column).  For  example, in Figure 9, A. firing elicited by Q U I S (393 spikes), L-GLU (383 spikes) and N M D A (293 spikes) is lower during the ejection of PDA; ie. a response reduction of 3 % (377 spikes-QUIS), 1 0 % (358 spikes-L-GLU) and 7 6 % (75 spikesN M D A ) occurred. The % reduction by PDA of firing elicited by each of the agonists is averaged for all the cells examined and is presented as the mean %  48  reduction plus or minus the standard deviation with the numbers of cells tested given in parentheses (Table 2, middle column). Secondly, the % reduction by an antagonist of firing elicited by an agonist is c o m p a r e d directly with the reduction of NMDA-elicited firing for each cell tested. This paired presentation in effect normalizes the d a t a to a standard agonist, N M D A . For example in Figure 9, A. the 3 % reduction of QUIS-induced activity by PDA is c o m p a r e d as a ratio to the 6 5 % reduction of NMDA-elicited firing to give 0.05.  The  difference between the reduction of Q U I S - and NMDA-elicited firing by P D A for this cell is greater than the average shown in Table 2, but clearly demonstrates the separation between these two agonists w h i c h , on s o m e but not all occasions, can be obtained with PDA. The specificity of PDA is moderate for NMDA-elicited firing a n d the degree of separation between that of Q U I S a n d N M D A with PDA d e p e n d s on achieving modest a n d stable responses to the agonists c o u p l e d with relatively low iontophoretic currents of PDA for extended periods. The first number in the right hand column of Table 2 for PDA indicates an average X / N M D A ratio for QUIS of 0.58.  This indicates that for a number of cells the reduction by P D A of the firing  induced by Q U I S will be on average 58 % of the reduction of NMDA-elicited activity. T h e s e ratios are averaged over the total number of cells on which an agonist w a s c o m p a r e d directly with N M D A , and are presented as the X / N M D A ratio plus or minus the standard deviation with the numbers of cells tested given in parentheses (Table 2, right hand column).  49  PDA  80 nA  Call A  o  o  •  386  414  326  153.  153  153.  o  o  Q  Q U I S 35 n A  O  L - G L U 60 n A  •  N M D A 40 nA  ( - ) L U L D J - C P A  72  I  153. '  'l53. '  o  A  •  317 i  282  351  O  A  •  20 3.  •  70  nA  108  229  i  '~0  A  •  1  K Y N A 50 n A  100  0  J  333  236  100  Figure 9.  Q  O U I S 28 nA  ^  K A I N 25 nA  Q  N M D A 30 n A  Segments of two continuous ratemeter records of the responses of single dorsal horn neurones in vivo to: cell A; QUIS (35 nA), L-GLU (60 nA) and N M D A (40 nA) and the antagonism of those responses by P D A applied iontophoretically at 80 nA and cell B; QUIS (28 nA), KAIN (25 nA) and N M D A (30 nA) and the antagonism of those responses by (-)trans-CPA and K Y N A applied iontophoretically at 70 and 50 nA respectively. Horizontal bars indicate the ejection period of compounds; the agonists being applied in an automatedtimed sequence and the antagonist currents being turned on and off manually. The total numbers of spikes elicited during each agonist ejection period are shown above each record.  50  Finally, the d a t a is presented in a tabular form in which the agonists are ranked in order of susceptibility to a given antagonist for each cell tested using a plus or minus 15 % distinction value. The first number of each pair in Table 3 represents those cells for which the firing elicited by the agonist at the head of the c o l u m n w a s reduced by at least 15 % more than w a s that of the agonist at the front of the row. T h e n u m b e r contained in parentheses indicates the n u m b e r of cells for which the two agonists could not be distinguished. This method of presentation allows each agonist to be c o m p a r e d directly to every other agonist for sensitivity to an antagonist. T h u s , the trends that can be seen at a glance are very helpful. For example, in Table 3 for PDA, the second pair of numbers from the top on the far right [11 (3)] indicate that for 11 cells on which both N M D A and Q U I S were tested with PDA, the NMDA-elicited firing w a s reduced by at least 15 % more than w a s that of Q U I S , while for three additional cells no distinction between N M D A and Q U I S could be made. T h e comparison between N M D A and Q U I S with PDA is made complete by examining the pair of numbers at the bottom of the second row from the left [0(3)], which indicates that firing elicited by Q U I S w a s never more sensitive to antagonism by PDA than w a s that induced by N M D A , while the same three cells are indicated for which no distinction between Q U I S a n d N M D A could be made. The conclusion, therefore, is, that of a total of 14 cells where the blockade of Q U I S and N M D A was directly c o m p a r e d using the antagonist PDA, on 11 of these N M D A - i n d u c e d activity was more potently attenuated than w a s that of Q U I S , while on three cells no distinction could be made.  51  Reduction of Amino Acid-Induced Activity of Dorsal Horn Neurones In vivo by Cis-2,3-piperidinedicarboxylate, Kynurenate a n d Acridinate  Agonist  % reduction of control firing  X / N M D A ratio  PDA QUIS  39.4 ± 1 7 . 7 (17)  0.58 ± 0 . 2 7 (14)  KAIN  49.6 ± 22.8 (8)  0.59 ± 0.23 (4)  NMDA  72.7 ± 16.7 (15)  1.00  QUIS  68.9 ± 2 4 . 9  (28)  0.70 ± 0 . 2 2 (24)  KAIN  75.9 ± 2 5 . 3  (18)  0.91 ± 0 . 1 3 (11)  NMDA  83.1 ± 2 1 . 6 (37)  1.00  QUIS  54.6 ± 1*9.8 (29)  0.82 ± 0.32 (23)  NMDA  69.7 ± 19.3 (26)  1.00  KAIN  73.7 ± 2 0 . 3  1.13 ± 0 . 3 2 (9)  KYNA  ACRA  Table 2.  (17)  Reduction of amino acid-induced firing of dorsal horn neurones in vivo by iontophoretically applied PDA, K Y N A and A C R A expressed as the % reduction of control firing ± S.D. Also, for each cell, the reduction of QUIS or KAIN induced firing by each of the antagonists is compared directly with the reduction of NMDA-induced activity and is expressed as the ratio: % reduction of X over the % reduction of N M D A ± S.D. (X / N M D A ratio). The numbers of cells tested are given in brackets. L-GLU was not tested on a sufficient number of cells to be included in this table, but does appear in Table 3.  52  Ranked Order of Paired Data for the Reduction of Amino Acid-Induced Activity of Dorsal Horn Neurones In vivo by Cis-2.3-piperidinedicarboxylate. Kynurenate a n d Acridinate  PDA L-GLU L-GLU  QUIS  KAIN  NMDA  3(0)  3(0)  2(0)  0(2)  11 (3)  QUIS  0(0)  KAIN  0(0)  1 (2)  NMDA  0(0)  0(3)  0(1)  QUIS  KAIN  NMDA  2(2)  3(3)  3(8)  10 (14)  L-GLU L-GLU  1 (3)  3(1)  QUIS  2(3)  KAIN  1 (2)  0(8)  NMDA  1 (3)  0(14)  0(8)  QUIS  L- G L U  NMDA  KAIN  12(9)  8(4)  3(8)  ACRA QUIS L-GLU  3(1)  NMDA  2(9)  KAIN  0(4)  3(4) 0(1)  Table 3.  2(4)  Antagonism of the amino acid-induced excitation of spinal neurones by PDA, K Y N A and A C R A in vivo . The figures in each column show the number of occasions on which excitation by the compound listed at the head of the column was selectively blocked when compared directly with the excitant listed at the left. Numbers in parentheses show the trials in which the two compounds could not be distinguished (±15 % ) . The excitants are listed from left to right, and from above downward in order of increasing susceptibility to blockade. A bar indicates that the two compounds were never directly compared.  53  The majority of the results a n d discussion to follow will deal with the paired X / N M D A ratios: decimal ratios will be given for agonists other than N M D A without referring constantly to the fact that they have been c o m p a r e d directly with N M D A , and that the reduction of agonist-induced activity has been normalized to N M D A = 1.00. Additional reference will be made to tables of ranked-paired d a t a in order to support conclusions, to indicate trends, or to make the groupings of the agonists more apparent.  B. Archetypal agonists a n d antagonism bv PDA. K Y N A a n d A C R A in the spinal cord in vivo In the first series of experiments, excitation of dorsal horn neurones by the archetypal agonists, Q U I S , KAIN a n d N M D A , a n d the reduction of these responses by two previously studied a n d one novel antagonist were e x a m i n e d in vivo.  Figure  9 s h o w s two ratemeter records of single dorsal horn neurones in vivo, demonstrating t h e actions of PDA, (-)trans-1-amino-1,2-cyclopentane dicarboxylate [ M t r a n s - C P A ] a n d KYNA. Cell A shows the reduction of Q U I S - (3 % ) , L-GLU(10 %) a n d N M D A - (65 %) elicited firing resulting from the application of PDA at 80 nA. T h e reduction of firing induced by QUIS (37 %) , KAIN (51 % ) a n d N M D A (96 %) by K Y N A applied at 50 nA is seen for cell B. Also shown for cell B is the reduction of agonist-induced firing by iontophoretically applied (-)trans-CPA which will be discussed in more detail later in this section. PDA, w h e n ejected at 40 to 80 nA from a 100 m M solution, w a s found to  54  reduce firing elicited by Q U I S , L-GLU and KAIN, but more potently attenuated activity due to NMDA. Table 2 shows that the ratios of the reduction of QUIS- and KAIN-elicited activities by PDA were 0.58 a n d 0.59 (ie. the reduction of Q U I S - and KAIN-elicited firing by any given iontophoretic dose of PDA w o u l d be on average 6 0 % that of N M D A - i n d u c e d acitivity). This finding concurs with results reported by Davies et al. (1981) for the cat spinal cord, although the separation between N M D A and Q U I S or KAIN appeared to be less in the cat. K Y N A w a s ejected at 25 to 70 nA from a 100 m M solution and w a s found to reduce KAIN a n d N M D A excitations comparably, while Q U I S activity w a s less affected. Direct comparison with N M D A s h o w e d activity induced by KAIN and Q U I S to be reduced 0.91 a n d 0.70 respectively (Table 2). These d a t a also are in general a g r e e m e n t with previous reports by Stone and Perkins (1984), G a n o n g et al. (1985) a n d Peet et al. (1986) in the hippocampus, Perkins a n d Stone (1984) in the cortex, Elmslie and Yoshikami (1985) in the frog spinal cord, a n d G a n o n g et al. (1983) in the rat spinal cord. The third antagonist tested in this series of experiments w a s acridinate (ACRA), a previously untested c o m p o u n d (synthesized by Dr. K. Curry). It was found to be less potent than KYNA and PDA, (ejected at 37 to 80 nA from a 200 m M solution) with little ability to distinguish between the three archetypal agonists. Table 2 shows that when c o m p a r e d directly with the reduction of NMDA-elicited firing, Q U I S and KAIN activities were altered to 0.82 a n d 1.13 respectively.  55  T h e ranked-paired data in Table 3 show that for 11 of 14 cells tested with PDA, N M D A - i n d u c e d firing w a s reduced by at least 15 % more than w a s Q U I S induced firing, while for 2 of 3 cells, Q U I S - and KAIN-activity w a s reduced by a similar amount. The agonists are shown from right to left and from the top d o w n in order of increasing susceptibility to antagonism. The trends apparent in Table 3 do not a p p e a r to contradict the conclusions gained from Table 2. T h u s , with PDA, the agonists appear to fall into two categories: (QUIS and KAIN) < N M D A .  Two  categories of agonists are also apparent w h e n using KYNA, however, KAIN is more like N M D A than Q U I S in its susceptibility to this antagonist, hence: Q U I S < (KAIN and NMDA).  C. Archetypal agonists and antagonism bv PDA. K Y N A and A C R A in the cortex in vivo In these experiments the excitation of cortical neurones in vivo by Q U I N , in addition to the archetypal agonists Q U I S , KAIN a n d N M D A , and the antagonism of these responses by iontophoretically applied K Y N A and A C R A were examined. Figure 10 s h o w s two ratemeter records from single cortical neurones in vivo which demonstrate for cell A the degree of separation between Q U I S - and NMDAelicited activity which can be achieved using KYNA, a n d for cell B, that responses to Q U I N a n d N M D A are often comparably reduced by K Y N A in the cortex. As in the spinal cord in vivo. A C R A (ejected at 50 to 80 nA from a 200 m M solution; Table 1: page 30) w a s found to be a relatively weak, nonspecific  56  KYNA  cell A. 278  50nA 271  89  165  246  239  spikes / sec.  A 111  15s.  10s.  o  o  • Q  •  i—i O  i—i •  Q U I S 30 nA r j  cell B.  KYNA  N M D A 1 3 nA  80nA  spikes / sec. 75  n  r^o  0 J 15 s.  20 s.  o  •  • Q  Q U I N 56 nA  •  o N M D A 1 9 nA  Figure 10. S e g m e n t s of t w o continuous ratemeter records of the responses of single cortical neurones in vivo t o : cell A; Q U I S (30 nA) a n d N M D A (13 nA) a n d the antagonism of those responses by K Y N A applied iontophoretically at 50 nA a n d cell B; Q U I N (56 nA) a n d N M D A (19 nA) a n d t h e antagonism of those responses by K Y N A applied iontophoretically at 80 nA. Conventions as in Figure 9 (page 50).  57  antagonist for which Q U I N , KAIN and Q U I S activity had reduction ratios of 0.85, 0.98 a n d 1.01 respectively (Table 4). Table 5 demonstrates using ranked-paired d a t a that for the majority of cells no distinction between amino acid agonists could be made using A C R A (± 15 % ) . W h e n ejected at 50 to 70 nA from a 100 m M solution (Table 1 : page 30), K Y N A reduced excitations elicited by all four agonists tested, with Q U I N , KAIN and N M D A being affected almost equally for most cells (0.96 a n d 1.13 for Q U I N and K A I N ; Table 4). QUIS-elicited activity w a s on average slightly less susceptible to antagonism by K Y N A in the cortex in vivo (0.77, Table 4). Table 5 shows that the agonists could be distinguished for a s o m e w h a t larger proportion of cells with K Y N A than with A C R A ; Q U I N , KAIN and N M D A appearing to fall into one group, with Q U I S the sole m e m b e r of a second group. T h e degree of separation between N M D A / Q U I N / K A I N and QUIS is not sufficient, however, to show a statistical difference between these two groups of c o m p o u n d s . T h e data presented so far for the spinal cord and cortex in vivo have been published in a slightly different form (Curry et al., 1986) and are in general agreement with results reported by many other authors (Davies et al., 1 9 8 1 ; M c L e n n a n and Liu, 1 9 8 1 ; Perkins and Stone, 1982; G a n o n g et al., 1983; M c L e n n a n , 1984; Elmslie and Y o s h i k a m i , 1985).  58  Reduction of Amino Acid-Induced Activity of Cortical Neurones In vivo by Kynurenate a n d Acridinate  Agonist  % reduction of control firing  X / N M D A ratio  KYNA QUIS  42.1 ± 16.6 (24)  0.77 ± 0 . 3 6 (18)  QUIN  45.5 ± 1 3 . 8 (12)  0.96 ± 0 . 4 9 (10)  NMDA  51.5 ± 17.1 (18)  1.00  KAIN  56.5 ± 2 3 . 0  1.13 ± 0 . 3 9  (12)  (12)  ACRA QUIN  40.3 ± 1 8 . 6 (8)  0.85 ± 0 . 2 5 (4)  KAIN  43.3 ± 1 5 . 8 (12)  0.98 ± 0 . 4 4 (11)  NMDA  48.9 ± 13.7 (13)  1.00  QUIS  41.7 ± 1 2 . 4 (20)  1.01 ± 0 . 3 2 (13)  Table 4.  Reduction of amino acid-induced firing of cortical neurones in vivo by K Y N A and A C R A expressed as the % reduction of control firing ± S.D. and the X / N M D A ratio as described for Table 2 (page 52). The numbers of cells tested are given in brackets.  59  Ranked Order of Paired Data for the Reduction of Amino Acid-Induced Activity of Cortical Neurones In vivo by Kynurenate and Acridinate  KYNA QUIS QUIS  QUIN  NMDA  KAIN  2(10)  6(11)  7(3)  5(4)  -  QUIN  1 (10)  NMDA  1 (11)  1 (4)  KAIN  1 (3)  -  2(6)  QUIN  QUIS  NMDA  KAIN  3(5)  1 (3)  1 (0)  2(10)  3(7)  4(6)  CRA QUIN QUIS  1 (5)  NMDA  0(3)  1 (10)  KAIN  0(0)  3(7)  Table 5.  4(4) 3(4)  Antagonism of the amino acid-induced excitation of cortical neurones by K Y N A and A C R A in vivo. Conventions are as described for Table 3 (page 53). The excitants are listed from left to right, and from above downward in order of increasing susceptibility to blockade. A bar indicates that the two compounds were never directly compared.  60  D. a-Substituted analogues in the spinal cord in vivo In this series of experiments substituted analogues of L-GLU were tested for agonist a n d antagonist properties in the spinal cord in vivo. a - M e t h y l , -phenyl and para-substituted-phenyl varieties were tested, but only the para-chloro ( a p C P G ) a n d para-fluoro ( a p F P G ; Fig. 1, F and G: page 3) substituted analogues had noticeable actions: these two were found to have weakly antagonistic effects. Table 6 shows the effects of a p C P G and a p F P G on dorsal horn neurone activity elicited by the iontophoretic application of N M D A , K A I N , Q U I S , L-GLU and A M P A in vivo. T h e analogue a p F P G reduced L-GLU-elicited firing more than that induced by N M D A . Table 7 demonstrates that for all 7 cells on which both L-GLU and N M D A were tested, the activity of L-GLU w a s reduced more than that of N M D A (± 15 % ) . All of the a-substituted analogues tested were ejected from 200 m M solutions (Table 1 : page 30) and a p F P G required iontophoretic currents averaging over 70 nA to cause any noticeable reduction of amino acid-induced firing.  E. (+) and (-) Trans-cyclopentane aspartate in the spinal cord in vivo T h e optical isomers of a previously untested analogue of aspartate were e x a m i n e d in these experiments for agonist or antagonist activities in the spinal cord in vivo. This c o m p o u n d , lraris_-1-amino-1,2-cyclopentane dicarboxylate (transcyclopentane aspartate; trans-CPA), was synthesized and resolved into its optical isomers by Dr. K. Curry. On no occasion did either isomer of trans-CPA cause excitation by itself or an enhancement of firing elicited by another c o m p o u n d ; on  61  Reduction of Amino Acid-Induced Activity of Dorsal Horn Neurones In vivo by oc-Substituted Analolgues of Glutamate  Agonist  ccpCPG  NMDA  gpFPG  -7.1 ± 1 9 . 8 (7)  KAIN  -15.0 ± 1 3 . 2 (3)  QUIS  -6.3±11.5(7)  AMPA  -2.7 ± 4 . 6 ( 3 ) -7.2 ±  n.t.  L-GLU  Table 6.  +19.1 ± 2 7 . 8 (11)  -6.5 ±  8.1(6)  -9.4 ± 1 7 . 9 (8) 7.5(4)  -23.7 ± 11.3 (12)  Effects of a-(para-chlorophenyl)glutamate and a-(para-fluorophenyl)-  glutamate on amino acid induced firing of dorsal horn neurones in vivo shown as the % change in control firing rate ± S.D. observed with the iontophoretic application of the a-substituted analogue, with the numbers of cells tested given in brackets.  Ranked Order of Paired Data for the Reduction of Amino Acid-Induced Activity of Dorsal Horn Neurones In vivo by cc-(para-fluorophenyl)glutamate  NMDA NMDA  KAIN  QUIS  AMPA  1 (1)  2(0)  2(0)  7(0)  -  -  2(0)  2(3)  3(2)  KAIN  0(1)  QUIS  0(0)  -  AMPA  0(0)  -  0(3)  L-GLU  0(0)  0(0)  0(2)  Table 7.  L-GLU  2(1) 1 (1)  Antagonism of the amino acid-induced excitation of cortical neurones by K Y N A and A C R A in vivo. Conventions are as described for Table 3 (page 53). The excitants are listed from left to right, and from above downward in order of increasing susceptibility to blockade. A bar indicates that the two compounds were never directly compared.  62  the contrary both were found to reduce amino acid-elicited firing in the dorsal horn. Figure 11 is a ratemeter recording of a single dorsal horn neurone in vivo showing the reduction of amino acid-induced activity by iontophoretically applied (-)trans-CPA.  W h e n applied at 50 nA from a 200 m M solution (Table 1: page 30),  (-)trans-CPA completely blocked the firing of this neurone elicited by Q U I S and L-GLU, c a u s e d a 90 % reduction of that induced by KAIN while reducing N M D A ' s activity by only 26 %. This pattern of antagonism can also be seen in Figure 9, B. (page 50) w h e r e (-)trans-CPA ejected at 70 nA c a u s e d 77 and 61 % reductions of Q U I S - and KAIN-activities respectively while reducing NMDA-elicited firing by only 37 %. W h e n tested on that s a m e neurone in Figure 9, B., K Y N A c a u s e d 37 and 51 % reductions of Q U I S - and KAIN-activities and almost completely blocked (96 %) the firing elicited by NMDA. Table 8 c o m p a r e s the antagonist activity exhibited by (+) and (-)trans-CPA on amino acid-induced firing of dorsal horn neurones. W h e n ejected at 25 to 90 nA from a 200 m M solution (Table 1: page 30) both isomers were capable of reducing firing elicited by all of the agonists tested. Q U I S , L-GLU and KAIN elicited activities were substantially more susceptible to antagonism by (-)trans-CPA than w a s N M D A - i n d u c e d firing. Paired students t-tests show that Q U I S , KAIN and L-GLU are not significantly different from one another in sensitivity to antagonism by (-)trans-CPA. but are significantly different from N M D A in that respect (p < 0.05). Table 9 supports these conclusions by demonstrating that for 15 of 16 neurones tested, the activity of QUIS was reduced more than that of N M D A by at least 15 %.  63  332  (-)trans-CPA  50nA  443  282  255  Figure 1 1 . Segments of a continuous ratemeter record of the responses of a single dorsal horn neurone in vivo to Q U I S (30 nA), L-GLU (40 nA), KAIN (35 nA) and N M D A (19 nA) and the antagonism of those responses by (-)trans-CPA applied iontophoretically at 50 nA. Conventions as in Figure 9 (page 50).  64  In addition, Table 9 shows that despite the statistical similarity, in 9 of 13 cells tested Q U I S - i n d u c e d firing w a s more sensitive to blockade by (-)trans-CPA than w a s that of KAIN ( ± 1 5 % ) .  F. Archetypal agonists and antagonism bv A P V . PDA. K Y N A and A C R A in the spinal cord in vitro All the remaining experiments to be discussed were done using the spinal cord slice preparation. The first goal w a s to examine many of the c o m p o u n d s which had already been tested in vivo in order to determine w h e t h e r or not their actions were similar in vitro. Table 10 shows the results for the first 30 cells from which records were obtained in the spinal cord in vitro and c o m p a r e s t h e m with the in vivo results already described. K Y N A a n d PDA were found to have similar profiles of antagonism in vitro as in vivo, in that the order of susceptibility of agonists to these antagonists w a s identical in the two preparations. T h e difference in the effect of K Y N A and PDA on excitations elicited by the most sensitive group of c o m p o u n d s a n d those of the least sensitive c o m p o u n d s is apparently greater in vitro than in vivo. For example, in Table 10, N M D A and KAIN excitations are the most sensitive of any tested to blockade by KYNA, while those of Q U I S are the least. W h e n e x a m i n e d in vivo the reduction ratios are 1.00 for N M D A , 0.91 for KAIN a n d 0.70 for Q U I S . The separation between QUIS and KAIN is, therefore, 0.19. W h e n studied in vitro, however, this separation grows to 0.30 (0.96 for KAIN and 0.66 for Q U I S ; Table 10). Similarly for PDA, both Q U I S - and KAIN-elicited  65  Reduction of Amino Acid-Induced Firing of Dorsal Horn Neurones In vivo by the Optical Isomers of Trans-1 -amino-1,2-cvclopentane dicarboxylate  (-)trans-CPA agonist  (+)trans-CPA  % reduction of control firing  % reduction of control firing  QUIS  73.4 ± 2 1 . 5 (24)  21.9 ± 24.2 (18).  L-GLU  68.6 ± 19.8 (13)  30.8 ± 34.2 (7)  KAIN  62.6 ± 2 1 . 9 (16)  39.4 ± 29.1 (11)  NMDA  29.8 ± 16.2 (20)  9.0 ± 2 4 . 4 (13)  Table 8.  Average iontophoretic current used: 63.5 and 72.3 nA respectively for (-) and (+Urans-CPA. Data given as the percent reduction of control firing resulting from iontophoretic application of the antagonists ± S.D. with the numbers of cells tested given in brackets.  Ranked Order of Paired Data for the Reduction of Amino Acid-Induced Activity of Dorsal Horn Neurones In vivo by (-)Trans-1-amino-1,2-cyclopentane  NMDA NMDA  dicarboxylate  KAIN  L-GLU  QUIS  4(4)  6(1)  15(0)  2 (7)  9 (2)  KAIN  0 (4)  L-GLU  0(1)  2(7)  QUIS  1 (0)  2 (2)  Table 9.  4(5) 1 (5)  Antagonism of the amino acid-induced excitation of spinal neurons by (-Urans-CPA in vivo. Conventions are as described for Table 3 (page 53). The excitants are listed from left to right, and from above downward in order of increasing susceptibility to blockade.  66  firing w a s less sensitive to blockade in vitro than in vivo relative to N M D A .  ACRA,  on the other hand, w a s as unspecific an antagonist in vitro as it w a s in vivo. Because PDA proved to have a profile of antagonism comparable to that reported for A P V , but with a lower potency and specificity for reducing NMDA-elicited firing c o m p a r e d to A P V (ie. the separation between N M D A and Q U I S a n d KAIN w a s not as great), A P V w a s used for the remaining experiments in addition to, a n d often to the exclusion of, PDA. During these first experiments using the spinal cord preparation in vitro it was discovered that 41 to 60 % lower agonist ejection currents were required to achieve similar firing rates w h e n c o m p a r e d to the situation in vivo. For Q U I S , N M D A and Q U I N the m e a n iontophoretic currents required to elicit a control firing rate of 15 to 25 Hz. were significantly lower in vitro than in vivo (p < . 0 0 1 , t-test; Table 11). For subsequent experiments involving KAIN and N M D A a lower concentration of agonist in the iontophoretic pipette w a s used in vitro c o m p a r e d to in vivo (Table 1 : page 30). This increase in potency was also found for antagonists, so that on average significantly lower iontophoretic currents were used in vitro to achieve reductions of Q U I S - or N M D A - i n d u c e d firing c o m p a r a b l e to those in vivo (66 % lower for PDA, 63 % lower for K Y N A ; p < . 0 0 1 , t-test; Table 11). These findings are shown in Table 1 1 , where the mean iontophoretic currents used to eject Q U I S , Q U I N , N M D A , KYNA and PDA are shown for many in vivo and in vitro experiments. The increased neuronal sensitivity in vitro meant that Q U I N , applied at 20 to 80 nA from a 200 m M solution (Table 1: page 30), w a s of sufficient potency  67  Reduction of A m i n o Acid-Induced Firing of Dorsal Horn Neurones In vivo and In vitro by Kynurenate, Acridinate, Cis-2.3-piperidine dicarboxylate and D-(-)-2-amino-5-phosphonovalerate  compound  QUIS  QUIN  KAIN  KYNA in vivo  0.70 ± 0 . 2 2 (17)  in vitro  0.66 ± 0.29 (16)  — 0.65 ± 0 . 1 7 (8)  0.91 ± 0 . 1 3 ( 1 0 0.96 ± 0.13 (11)  ACRA in vivo  0.82 ± 0 . 3 2 (21)  in vitro  1.13 ± 0 . 3 9 (11)  — 1.02 ± 0 . 4 9 (7)  1.13 ± 0 . 3 2 (9) 1.10 ± 0 . 3 7 (7)  PDA in vivo  0.58 ± 0 . 2 7 (14)  —  0.59 ± 0.23 (4)  in vitro  0.49 ± 0.34 (7)  —  0.51 ± 0.43 (6)  —  0.22 ± 0.28 ( 3 3 )  APV in vivo  0.37 ± 0.23 ( 4 6 )  invitro  0.23 ± 0.21 (14)  1  0.45±0.34(7)  Table 10.  1  0.18±0.18(7)  For each cell the effect of iontophoretically applied KYNA, A C R A , P D A and A P V has been expressed as the X / N M D A ratio as described for Table 2 (page 52). The numbers of cells tested are given in brackets. QUIN was not able to excite cells sufficiently to be tested in vivo. The effect of P D A on QUIN-induced firing was not examined. 1 -data from experiments by H. McLennan.  68  to be e x a m i n e d in this preparation; although of all the cells which were excited by Q U I S a n d N M D A , only 60 - 70 % would respond adequately to Q U I N . This is in contrast to its failure to excite more than a very small proportion (< 10 %) of spinal neurones in vivo (observations of the author a n d cf. Stone a n d Perkins, 1 9 8 1 ; M c L e n n a n , 1984). T h e s e initial experiments s h o w e d that the neurones being e x a m i n e d in the spinal cord slice respond to the amino acid agonists and antagonists in a manner qualitatively similar to what had been observed in vivo. They also d e m o n s t r a t e d that Q U I N ' s action in the spinal cord in vitro is unlike that of N M D A , in that its blockade by K Y N A w a s significantly less than that of N M D A (Table 10). This is in contrast to what has already been described herein for the actions of N M D A and Q U I N in the cortex w h e r e they were comparable in their sensitivity to blockade by K Y N A (Tables 4 and 5: pages 59 and 60). Q U I N has been reported by others to be qualitatively indistinguishable from N M D A in the cortex (Perkins and Stone, 1982; M c L e n n a n , 1984), a n d h i p p o c a m p u s (Ganong and C o t m a n , 1986; Peet et al., 1986).  69  C o m p a r i s o n of Agonist and Antagonist Ejection Currents Used in the Spinal Cord In vivo & In vitro  compound  QUIS  In Vivo (nA) (5mM)  22.4 + 9 . 9 ( 1 0 9 )  mean firing rate  20.1 Hz  NMDA (50mM)  30.8 ± 14.6 (117)  mean firing rate  QUIN  19.4 Hz  (200mM)  94.0 ± 2 3 . 0 (5)  mean firing rate  KYNA  18.4 Hz  (20mM)  51.0 ± 1 0 . 6 (26)  mean firing rate reduction PDA  68.1 %  (100mM)  54.0 ± 11.3 (14)  mean firing rate reduction  68.7 %  In Vitro (nA) 12.5 + 8 . 7 ( 5 1 ) 19.1 Hz 18.2 ± 10.4 (94) 18.9 Hz 38.1 ± 16.9 (41) 19.9 Hz 34.2 ± 16.3 (36) 74.2 % 19.8 ± 8 . 4 (18) 67.5 %  In each case the firing rate achieved with the degree of reduction induced is not significantly different in vivo vs in vitro. The mean currents needed to elicit the effects however were always less in vitro (p < 0.001, t-test).  Table 1 1 .  QUIS, N M D A and QUIN: The average iontophoretic current (± S.D.) required to evoke a control firing rate of between 15 and 25 Hz. K Y N A and PDA: The average iontophoretic current required to achieve a 50 to 8 0 % reduction of QUIS- or NMDAinduced firing. The numbers of cells tested are given in brackets.  70  G. L- a n d D-glutamate in the spinal cord in vitro Both the L- a n d D-isomers of glutamate were e x a m i n e d on a n u m b e r of cells using the spinal cord preparation in vitro. T h e s e results are shown as the pooled % reduction a n d paired X / N M D A ratios in Table 12 and as ranked-order paired d a t a in Table 13. Paired t-tests determined that the blockade of D-GLU-induced activity w a s significantly greater than that of Q U I S but significantly less than that of N M D A for both A P V a n d KYNA (paired t-tests; Table 12). In contrast, no significant difference could be s h o w n between the reductions of Q U I S - a n d L-GLU-elicited firing by either A P V or by KYNA. This grouping, with L-GLU a n d Q U I S being the least, D-GLU intermediate a n d N M D A being the most sensitive to antagonism by A P V and K Y N A can also be seen in Table 13. Most notably, D-GLU-elicited firing w a s more sensitive (± 15 %) to antagonism by both A P V a n d K Y N A than were those of Q U I S a n d L-GLU on all cells examined (top two pairs of numbers in the third c o l u m n of Table 13 for A P V and KYNA). That D-GLU is not purely NMDA-like w a s also s e e n , however, since for 8 of 9 and 3 of 5 cells e x a m i n e d , it w a s less sensitive to antagonism by A P V and K Y N A respectively than w a s N M D A (Table 13). W h e n the iontophoretic currents required to evoke a similar firing rate from a given neurone were c o m p a r e d for these agonists, Q U I S w a s found to be 43 times and D-GLU 0.43 times as potent as L-GLU (Table 12). T h e s e conclusions are in agreement with earlier reports on the activity of D-GLU (Hicks et al., 1978; Hall et al., 1979).  71  R e d u c t i o n of A m i n o A c i d - I n d u c e d Activity of Dorsal Horn N e u r o n e s In vitro by D - ( - ) - 2 - a m i n o - 5 - p h o s p h o n o v a l e r a t e compound  potency*  and Kynurenate  a v e r a g e % reduction  X / N M D A (paired)  APV D-GLU  0.43  5 6 . 6 + 18.1 (10)  0) 0.64 ± 0.15 (9)  L-GLU  1.00  2 3 . 4 ± 14.3 (10)  ( ) 0.26 ± 0.17 (10)  QUIS  43.0  23.1 ± 17.7 (15)  (b) 0.29 ± 0.21 (12)  D-GLU  4 6 . 2 ± 15.7 (5)  ( ) 0.70 ± 0.24 (5)  L-GLU  3 0 . 3 ± 16.9 (7)  (*>) 0.48 ± 0.26 (6)  QUIS  32.5 ± 1 5 . 7 (15)  (*» 0.51 ± 0.24 (10)  b  KYNA  a -significantly different from QUIS and NMDA. b -significantly different from NMDA.  a  paired t-tests, p < 0.05. paired t-tests, p < 0.005.  T a b l e 12. Reduction of amino acid-induced firing of dorsal horn neurones in vitro by K Y N A and A P V expressed as the % reduction of control firing ± S.D. and as the X / N M D A ratio as described for Table 2 (page 52). The numbers of cells are given in brackets. 'Potency: agonist potency compared to L-GLU = 1.00. For example, D-GLU requires about 2.3 X the ejection current needed by L-GLU to achieve a similar effect on a given neurone, making allowance for the total ionic strength of the solutions.  72  Ranked Order of Paired Data for the Reduction of Amino Acid-Induced Activity of Dorsal Horn Neurones In vitro by D-(-)-2-amino-5-phosphonovalerate a n d Kynurenate  APV QUIS QUIS  L-GLU  D-GLU  NMDA  0(2)  2(0)  12(0)  8(0)  10(0)  L-GLU  0(2)  D-GLU  0(0)  0(0)  NMDA  0(0)  0(0)  0(1)  QUIS  L-GLU  D-GLU  NMDA  0(2)  2(0)  9(1)  4(0)  5(1)  8(1)  'NA QUIS L-GLU  1 (2)  D-GLU  0(0)  0(0)  NMDA  0(1)  0(1)  Table 13.  3(2) 0(2)  Antagonism of the amino acid-induced excitation of dorsal horn neurones in vitro by A P V and KYNA. Conventions are as described for Table 3 (page 53). The excitants are listed from left to right, and from above downward in order of increasing susceptibility to blockade.  73  H. Quinolinate in the spinal cord in vitro: antagonists applied iontophoretically and topically Figure 12 shows typical segments of a continuous ratemeter record which c o m p a r e s the reduction of amino acid-induced firing in a single lamina IV neurone in vitro by A P V a n d A C R A applied iontophoretically or via the superfusate, and by K Y N A applied in the superfusate. For this cell APV applied iontophoretically at 9 nA reduced N M D A - e v o k e d firing (94 % ) , had a minimal effect on the response elicited by Q U I S (26 % ) , a n d had an intermediate effect on Q U I N - i n d u c e d firing (51 % ) . Similarly, APV applied in the superfusate at 4 x 1 0 " M abolished the 6  response to N M D A (100 %) while reducing the QUIS-elicited firing 45 % and that of Q U I N 60 % (Fig. 12).  KYNA w a s less specific than A P V , thus, w h e n topically  applied at 5 x 1 0 " M , firing elicited by N M D A was reduced 85 % while that of Q U I S 5  and Q U I N w a s reduced 57 and 61 % respectively. T h e nonspecific antagonist A C R A applied at 4 x 1 0 ' M caused approximately a 5 0 % reduction of the activities 4  elicited by all three agonists. In Table 14, the reduction of amino acid-induced firing of dorsal horn neurones in vitro by iontophoretically applied antagonists is s h o w n , as it w a s in part in Table 10. For each cell examined, the reduction of QUIS-, Q U I N - and KAINinduced firing, expressed as a percent of control, is c o m p a r e d with the reduction of NMDA-elicited activity.  Responses to Q U I S and KAIN are largely unaffected by  A P V (0.23 a n d 0.18 respectively) at ejection currents which reduced N M D A - e v o k e d responses on average by 9 3 % (n = 18). The antagonism of QUIN-activity by APV  74  O  A  •  O  A  •  O  A  •  Figure 12. S e g m e n t s of a continuous ratemeter record of the responses of a single dorsal horn neurone in vitro to QUIS (15 nA), Q U I N (95 nA) and N M D A (44 nA) and the antagonism of those responses by: APV applied iontophoretically at 9 nA and in the superfusate at 4 u.M, A C R A applied iontophoretically at 50 nA and in the superfusate at 400 JIM and K Y N A applied in the superfusate at 50 u,M. Conventions as in Figure 9 (page 50). Stock solutions of the antagonists were diluted in 10 ml. of gased A C S F and applied at the standard superfusion rate of 1.5 ml.- min.- . 1  75  Reduction of Amino Acid-Induced Activity of Dorsal Horn Neurones In vitro by D-(-)-2-amino-5-phosphonovalerate, Kynurenate a n d Acridinate  Agonist  % reduction of control firing  X / N M D A ratio  APV KAIN  18.5 ± 1 6 . 3  (10)  0)0.18 ± 0 . 1 8  (7)  QUIS  20.1 ± 1 9 . 1  (19)  ( >0.23 ± 0 . 2 1  (14)  QUIN  47.1 ± 3 1 . 0  (11)  (2)0.45 ± 0 . 3 4  (7)  NMDA  93.0 ±  8.6 (16)  1  ( >1.00 3  KYNA QUIN  53.0 ± 26.8 (14)  (2)0.65 ± 0 . 1 7  (8)  QUIS  57.7 ± 3 6 . 2  (27)  (2)0.66 ± 0 . 2 9  (16)  KAIN  81.7 ± 25.9 (15)  ( )0.96 ± 0 . 1 3  (11)  NMDA  87.3 ± 2 2 . 0  (30)  ( )1.00  QUIN  64.1 ± 2 2 . 4  (9)  NMDA  71.5 ± 24.0 (15)  1.00  KAIN  79.5 ± 2 6 . 8  (6)  1.10 ± 0 . 3 7  (7)  QUIS  69.5 ± 3 2 . 0  (12)  1.13 ± 0 . 3 9  (11)  3  3  ACRA  (1) significantly different from QUIN and N M D A (2) significantly different from KAIN and NMDA (3) significantly different from QUIN and QUIS  1.02 ± 0 . 4 9  (7)  paired t-tests (p<0.05)  Table 14. Reduction of amino acid-induced firing of dorsal horn neurones in vitro by iontophoretically applied APV, K Y N A and A C R A expressed as the % reduction of control firing ± S.D. and as the X / N M D A ratio as described for Table 2 (page 52). The numbers of cells tested are given in brackets.  76  w a s found to be 0.45 averaged for 7 cells, which w a s significantly greater than that of Q U I S - and KAIN-induced firing but significantly less than that of N M D A (p<.001,t-test). Using A P V as an antagonist, the agonists can be separated into three significantly different groups with Q U I S a n d KAIN the least susceptible, Q U I N intermediately affected and N M D A the most sensitive to blockade. In contrast, iontophoretically applied K Y N A caused similar reductions of Q U I S - a n d Q U I N e v o k e d responses (0.66 and 0.65 respectively), while KAIN-elicited firing could not be distinguished from N M D A (0.96 c o m p a r e d with N M D A = 1.00). Thus, only two significantly separable categories are apparent w h e n using K Y N A : Q U I S a n d Q U I N are the least affected while KAIN and N M D A are much more susceptible. Reductions of agonist-induced firing by A C R A were similar for all four of the agonists studied a n d no statistical differences could be s h o w n for any of the excitatory c o m p o u n d s tested. Figure 13 shows the dose-response curves for K Y N A and A P V applied in the bathing m e d i u m . The concentration range used for K Y N A is precisely 10-fold larger than that of A P V which gives a good indication of the relative potencies of these two antagonists against N M D A activity. The blockade of Q U I N , Q U I S , and KAIN-induced firing is incomplete at 20 u.M APV however, and the slopes of the curves at this concentration suggest that doses of A P V several fold higher would have to be used to block completely the actions of these agonists. In contrast, the highest dose of K Y N A shown in Figure 13 of 200 u.M was sufficient to abolish the  77  Concentration  Concentration  of A P V  (JLIM)  of K Y N A  (pM)  Figure 13. Dose-response curves for the actions of kynurenate (KYNA; bottom) a n d D-(-)-2-amino-5-phosphonovalerate (APV; top) against excitations elicited by the iontophoretic application of Q U I S , Q U I N , KAIN and NMDA. Points on the middle part of each curve are means of a minimum of 3 cells. The standard deviation is indicated for one point on each curve.  78  activity of all the agonists on virtually every neurone e x a m i n e d . Furthermore, the larger separation between the A P V dose-response curves for the various agonists d e m o n s t r a t e s the greater separation of effects which can be achieved with A P V c o m p a r e d to KYNA. Table 15 contains the IC50's determined directly from the d o s e - r e s p o n s e curves which represents the average concentration of antagonist required to reduce agonist-induced firing by 5 0 % . T h e Figure 13 potency orders d e t e r m i n e d from the graph are identical to those calculated from, the iontophoretic d a t a ( N M D A = KAIN) > (QUIS = QUIN) for K Y N A and N M D A > Q U I N > (QUIS = KAIN) for A P V .  Action of Perfusate Applied Antagonists:  Antagonist  KYNA  APV  Agonist  Cells Tested  IC50's  IC50 (p.M)  QUIS  19  120.0  QUIN  15  120.0  KAIN  14  50.0  NMDA  19  40.0  QUIS  17  18.0  QUIN  18  7.0  KAIN  10  20.0  NMDA  18  2.0  Range (6 - 200 uJvl)  (0.5-20U.M)  T a b l e 15. IC50's were determined directly from the dose-response curves for the two antagonists. Cell numbers are totals for all concentrations used. Note that the majority of cells were tested at more than a single antagonist concentration.  79  T h e s e results can be seen as ranked order of paired d a t a in Table 16. This Table shows that for 6 of 7 a n d 8 of 8 cells respectively, the action of N M D A was more sensitive to A P V and K Y N A than w a s that produced by Q U I N . In addition, for 6 of 11 cells tested with A P V , QUIN-activity w a s reduced more than w a s Q U I S induced firing while the converse never occurred. O n several occasions the normal bathing m e d i u m w a s replaced with one in which additional sodium ions were substituted for the normal concentration of m a g n e s i u m ions ( M g ) . The reduction of NMDA-elicited firing by physiological 2 +  levels of M g  2 +  (Davies a n d Watkins, 1977) w a s thereby r e m o v e d , which resulted in  a two-fold increase in the size of N M D A - i n d u c e d responses. Table 17 shows that for six of the eight cells studied with M g - f r e e m e d i u m , Q U I N - r e s p o n s e s were 2+  c o m p a r e d directly with those of N M D A a n d found to increase an average of 104 %. Firing elicited by Q U I S w a s also examined for 7 of the 8 cells tested with N M D A and were observed to increase an average of 42 % giving an X / N M D A ratio of 0.52. Paired t-tests disclosed that Q U I N and N M D A responses increased comparable a m o u n t s in M g - f r e e medium (p > 0.375), while those of Q U I S were increased a 2+  significantly smaller amount (p < 0.05). Despite the ability of A P V and K Y N A to distinguish between the activities of Q U I N and N M D A in the dorsal horn in vitro, removal of M g  2 +  from the medium does not do so.  80  Ranked Order of Paired Data for the Reduction of Amino Acid-Induced Activity of Dorsal Horn Neurones In vitro by D-(-)-2-amino-5-phosphonovalerate, Kynurenate a n d Acridinate  APV KAIN QUIS  2(8)  NMDA 6(5)  14(0)  1 (2)  7(0)  KAIN  1 (8)  QUIN  0(5)  0(2)  NMDA  0(0)  0(0)  0(1)  QUIS  QUIN  KAIN  NMDA  1 (5)  5(1)  13(0)  4(1)  8(0)  6(1)  'NA QUIS QUIN  2(5)  KAIN  0(1)  0(1)  NMDA  0(0)  0(0)  1 (4)  QUIS  QUIN  KAIN  2(4)  ACRA 2(4)  QUIS QUIN  2(4)  KAIN NMDA  3(3) 0(2)  2(2) 2(3)  1 (6)  Table 16.  NMDA  1 (6) 1 (3)  2(3)  Antagonism of the amino acid-induced excitation of spinal neurones, in vitro, by APV, K Y N A and A C R A . Conventions are as described for Table 3 (page 53). The excitants are listed from left to right, and from above downward in order of increasing susceptibility to blockade. A bar indicates that the two compounds were never directly compared.  81  The Effect of Magnesium-Free Bathing Medium on Dorsal Horn Neurone Firing Elicited by Quisqualate, Quinolinate a n d N M D A In vitro  QUIS  QUIN  NMDA  A. % increase  42 ± 3 7 (8)  104 ± 5 8 (6)  102 ± 6 3 (8)  B. X / N M D A  0.52 ± 0 . 2 2 (7)  1.04 ± 0 . 2 0 (6)  1.00  Table 17.  Data presented in line A as the percent increase in agonist-elicited firing in response to magnesium-free bathing medium (+ S.D.) and in line B as the X / N M D A ratio as described for Table 2 (page 52). The numbers of cells tested are given in brackets. Paired t-tests show QUIS to be significantly different from both QUIN and N M D A (p < 0.005) while QUIN and N M D A are not significantly different (p > 0.375).  I. C o n f o r m a t i o n a l ^ restricted analogues: 1-amino-1.3-cvclopentane dicarboxylate a n d pyridine dicarboxylate The final series of experiments examined the actions of the optical and geometric isomers of 1-amino-1,3-cyclopentane dicarboxylate (ACPD) a n d several derivatives of pyridine closely related to Q U I N . The isomers of A C P D were first e x a m i n e d in the in vivo preparation a n d were found to be excitants. They were then studied in vitro using primarily iontophoretically applied A P V a n d K Y N A as antagonists, but several experiments using topically administered antagonists were done to confirm the iontophoretic results. On no occasion were discrepancies detected.  82  J. C o m p o u n d s acting at N M D A receptors Figure 14 shows a series of ratemeter records of the firing of single dorsal horn neurones. For cells A and B, firing elicited by N M D A is c o m p a r e d directly with that of two A C P D isomers for susceptibility to iontophoretically applied A P V and K Y N A and topical APV. For cell A, the activities elicited by D-cis- a n d L-cis-ACPD and N M D A were reduced similarly by A P V applied at 3 nA (ca. 85 % ) . Topical APV also resulted in reductions of similar magnitudes for these three agonists (ca. 55 % ) . In contrast, firing induced by D-trans-ACPD  remained at more than  60 % of control (less than a 40 % reduction) during the iontophoretic application of K Y N A at 10 nA, a dose which, on this particular cell, completely blocked the action of D-c]s.-ACPD a n d reduced the firing resulting from N M D A by ca. 75 %. The results obtained with all of the isomers of A C P D are included in Table 18 for A P V a n d Table 20 for KYNA, a n d a comparison of their potencies with those of many other agonists is also found in Table 18. W h e n ejection currents required to evoke a similar level of firing in a number of neurones were c o m p a r e d , D-cis-. L-trans-. a n d L-cis-ACPD were 1.8, 0.58 and 0.32 times as potent as L-GLU respectively, while N M D A w a s found to have a potency ratio of 5 (Table 18). W h e n c o m p a r e d to N M D A , therefore, D-cis-. L-trans-. and L - d s - A C P D were found to be 3, 9 and 16 times less potent.  The pattern of potency observed for these  c o m p o u n d s a p p e a r e d to parallel their sensitivity to A P V ; L-trans- and particularly L-cis-ACPD often retained a small degree of residual activity in the presence of doses of A P V or K Y N A which completely blocked N M D A and D-c]s_-ACPD.  83  APV  Cell A. spikes / sec  173  228  218  3nA 37  29  30  50-,  1  I  A  1  D APV  187  202  173  1  I  O 8nM 91  76  83  50-,  OJ ~l  A  I  I  D  1  I  O 169  174  190  50-,  OJ  1  •  o  261  292  360  15S.  15S.  A  •  15 s.  207  234  l  A  £±  D- c J i - A C P D  [~]  N M D A 1 6 nA  O  L-LL2-ACPD  KYNA  Cell B. spikes/ sec. 50.,  1 4 nA  1 8 nA  10 n A 216  69  OJ  A  o 342  1  I  i — i  •  O  ^  D-CjJS-ACPD  1 4 nA  Q  N M D A 1 6 nA  <0  D- I r a n s - A C P D  50^  I  I —I -  A  D  1  23 nA  I 1  O  Figure 14. S e g m e n t s of two continuous ratemeter records of the responses of single dorsal horn neurones in vitro to: Cell A. D-cjs.-ACPD (14 nA), N M D A (16 nA) and L-cis-ACPD (18 nA) and the antagonism of those responses by: APV applied iontophoretically at 3 nA and in the superfusate at 8 u.M. Cell B. D-cis-ACPD (14 nA), N M D A (16 nA) and D-trans-ACPD (23 nA) and the antagonism of those responses by K Y N A applied iontophoretically at 10 nA. Conventions as in Figure 9 (page 50).  84  Reduction of A m i n o Acid-Induced Activity of Dorsal Horn N e u r o n e s In vitro by D - ( - ) - 2 - a m i n o - 5 - p h o s p h o n o v a l e r a t e  compound  potencv*  averaae % reduction  X / N M D A toaired) 1.00  NMDA  5.0  97.1 ± 5 . 5 (20)  D-ds-ACPD  1.8  96.8 ± 6 . 7  L-trans-ACPD  0.58  72.5 ± 13.6 (10)  (a) 0 . 9 2 ± 0 . 1 6 ( 1 0 )  L-cis-ACPD  0.32  75.9 ± 19.4 (10)  (a) 0 . 8 7 ± 0 . 1 8 ( 1 0 )  D-trans-ACPD  0.25  45.7 ± 2 6 . 1 (7)  (b) 0.51 ± 0 . 2 6 ( 7 )  QUIN  0.24  47.3 ± 2 5 . 0 (14)  (b) 0 . 4 5 ± 0 . 3 4 (7)  L-GLU  1.00  23.4 ± 14.3 (10)  (b, d) 0 . 2 6 ± 0 . 1 7 ( 1 0 )  QUIS  43  20.3 ± 16.0 (20)  (c) 0 . 2 3 ± 0.21 ( 1 4 )  2,6-PyrDA  0.27  17.6 ± 2 2 . 6 (10)  (c) 0 . 2 2 ± 0 . 2 5 ( 1 0 )  2,5-PyrDA  0.19  18.1 ± 1 3 . 9 (9)  (9)  (a) 1.00 ± 0 . 0 3 (9)  •(c) 0 . 1 9 ± 0 . 1 5 (8)  a -not significantly different from NMDA. paired t-tests p > 0.375. b-significantly different from NMDA. paired t-tests p < 0.05. c -significantly different from NMDA and QUIN. paired t-tests p < 0.05. d -significantly different from QUIN unpaired t-test p < 0.05  Table 1 8 . Reduction of amino acid-induced firing of dorsal horn neurones in vitro by A P V *  expressed as the % reduction of control firing ± S.D. and as the X / N M D A ratio as described for Table 2 (page 52). The numbers of cells tested are given in brackets. potency: compared to L-GLU = 1.00. For example, both D-trans-ACPD and QUIN require 4 X the ejection current needed by L-GLU to achieve a similar effect on a given neurone, making allowance for the total ionic strength of the solutions. Not shown is KAIN = 20. When compared directly with NMDA, KAIN is approximately 4 X more potent, thus making it 20 X the potency of L-GLU.  85  T h e difference between the susceptibility of L-trans-. L-cjs-ACPD and N M D A to blockade by A P V is not statistically significant using paired t-tests (p > 0.375). T h e s e d a t a a p p e a r as ranked order of paired results in Table 19, which clearly illustrates that D-cis-. L-cis- and L-trans-ACPD are NMDA-like. In the top right quadrant of the table for A P V , which shows the results w h e n these c o m p o u n d s were tested against Q U I S , Q U I N or D-trans-ACPD. the NMDA-like c o m p o u n d s were more strongly affected by A P V in 34 of 36 cells (± 15 % ) . The bottom right quadrant of this table, where these c o m p o u n d s were c o m p a r e d against one another, shows that a distinction w a s made with A P V between two m e m b e r s of this g r o u p only 7 times out of 61 trials, and all 7 of those experiments were when D-cis-ACPD or N M D A were c o m p a r e d directly with L-cis- or L-trans-ACPD. c o m p o u n d s found to retain s o m e residual activity in the presence of antagonist. Table 20 s h o w s the d a t a obtained while using iontophoretically applied KYNA. Activities elicited by D-cis-. L-cis- and L-trans-ACPD were consistently reduced to a comparable extent by KYNA, a n d no statistical differences could be s h o w n between any of these c o m p o u n d s and N M D A . T h e s e d a t a appear as ranked order of paired results in Table 19. It can be seen in this table that although K Y N A w a s unable to distinguish between members of this group in 42 of 44 trials (bottom right quadrant), a distinction w a s made in 29 out of 30 experiments during which these c o m p o u n d s were tested directly with KYNA against Q U I S , Q U I N or D-trans-ACPD.  86  Ranked Order of Paired Data for the Reduction of A m i n o Acid-Induced Activity of Dorsal Horn Neurones In vitro by D-(-)-2-amino-5-phosphonovalerate a n d K y n u r e n a t e  APV QUIS QUIS  QUIN  DT  -  1 (0) 0(2)  LC  LT  DC  NMDA  I  3(0)  6(0)  3(0)  11 (0)  |  -  -  -  3(1)  -  -  2(0)  6(1)  1 (1)  1 (2)  3(7)  -  2(8)  QUIN  -  DT  0(0)  1 (2)  LC  0(0)  -  |  LT  0(0)  -  |  0(1)  DC  0(0)  -  0(0)  |  0(2)  -  NMDA  0(0)  0(1)  0(1)  I  0(7)  0(8)  0(9)  QUIS  QUIN  DT  LC  LT  DC  NMDA  -  0(0)  |  2(0)  4(0)  3(0)  9 (0)  0(2)  |  -  -  -  2(1)  -  1 (0)  2(0)  6(0)  0(4)  -  0(4)  -  2(7)  QUIS QUIN  -  DT  1 (0)  0(2)  LC  0(0)  -  -  LT  0(0)  -  0(0)  DC  0(0)  -  NMDA  0(0)  0(1)  •0(0) 0(0)  Table 19.  |  0(4)  |  -  -  |  0(4)  0(7)  0(9)  0(6) 0(6)  Antagonism of the amino acid-induced excitation of dorsal horn neurones in vitro by A P V and KYNA. Conventions are as described for Table 3 (page 53). The excitants are listed from left to right, and from above downward in order of increasing susceptibility to blockade. A bar indicates that the two compounds were never directly compared. DT, LC, LT and D C represent D-trans-. L-cis-. L-trans- and D-cis-ACPD respectively.  87  K. C o m p o u n d s acting at QUIS receptors Of all the conformationally restricted c o m p o u n d s tested only 2,5- and 2,6-PyrDA elicited activity which was pharmacologically similar to that of L-GLU a n d Q U I S . W h e n these c o m p o u n d s were c o m p a r e d with Q U I S - , K A I N - and NMDAinduced firing for susceptibility to blockade by A P V and K Y N A , activity evoked by 2,5- a n d 2,6-PyrDA w a s resistant to blockade by A P V (Table 18, Figs. 15 and 16) a n d only moderately sensitive to K Y N A (Table 20, Figs. 15 a n d 16). Figures 15 and 16 are ratemeter records of single dorsal horn neurones recorded in vitro. In Figure 16, cell A., activity induced by Q U I S w a s 18 % lower a n d that by 2,6-PyrDA 16 % lower than control firing during the iontophoretic application of APV at 9 nA while NMDA-elicited activity is reduced by 50 %. A qualitatively similar result is found in Figure 16, cell B., where Q U I S and 2,6-PyrDA activities were reduced by approximately 50 % while those of N M D A were almost completely blocked w h e n KYNA w a s applied at 7 nA. That cell activity elicited by Q U I S and 2,5-PyrDA is less susceptible to antagonism by A P V and K Y N A than that of N M D A is shown in Figure 15. W h e n tested with A P V applied at 2 nA (cell A) Q U I S - and 2,5-PyrDA-activities were reduced by 40 and 5 % respectively while the action of N M D A was lowered by 94 %. For cell B., KYNA applied at 5 nA resulted in a 22 % reduction of Q U I S - and a 6 % reduction of 2,5-PyrDA-elicited firing while that induced by N M D A decreased by 68 %. The pyridine analogues 2,5- and 2,6-PyrDA were the least susceptible to blockade by A P V of all the conformationally restricted analogues tested and in this  88  APV 106  I  O  197 50  202  193  -,  0  O  A  157  192  spikes/sec.  O  I 1  A  0  •  O  J  A  A  I  2 , 5 - P y r D A 48 nA  •  NMDA40nA  KYNA  -,  o  l  A  205  158 50  I  QUIS 1 4 nA  123  1  I  •  50 _,  I  11  180  Q  D  J  2nA  5nA 60  172  i  A  i •  Q  QUIS 1 4 n A  A  2,5-PyrDA  O  N M D A 40 nA  48 nA  •  Figure 15. Segments of two continuous ratemeter records of the responses of single dorsal horn neurones in vitro t o : Cell A. QUIS (4 nA), 2,6-PyrDA(24 nA) and N M D A (12 nA) and the antagonism of those responses by: A P V applied iontophoretically at 9 nA, and cell B. Q U I S (5 nA), 2,6-PyrDA (13 nA) a n d N M D A (12 nA) a n d the antagonism of those responses by K Y N A applied iontophoretically at 7 nA. Conventions as in Figure 9 (page 50).  89  Cell A spikes /sec.  362  267  APV  267 224  50 _.  0  298  130  J  281  349  256  50  0  9nA  -  Q  QUIS 4 n A  / \  2 , 6 - P y r D A 24 n A  Q  NMDA  1 2 nA  Cell B. spikes/sec.  214  220  K Y N A 7 nA  276 97  50 _.  109  A  /  0 _ 15 s.  15 s.  O  A  191  187  50 _,  0  15 s.  •  o  233  A  J  I  O  1  r-—  A  1  I  i  i  A  Q  Q Ul S  / \  2 , 6 - P y r D A 1 3 nA  Q  NMDA  i — i •  5 nA  21 nA  •  Figure 16. Segments of two continuous ratemeter records of the responses of single dorsal horn neurones in vitro t o : Cell A. Q U I S (14 nA), 2,5-PyrDA (48 nA) and N M D A (40 nA) a n d the antagonism of those responses by A P V applied iontophoretically at 2 nA, a n d cell B. QUIS (4 nA), 2,5-PyrDA (48 nA) a n d NMDA (40 nA) and the antagonism of those responses by K Y N A applied iontophoretically at 5 nA. Conventions as in Figure 9 (page 50).  90  respect are indistinguishable from L-GLU and Q U I S , although less potent (potency ratios 0.27 and 0.19 c o m p a r e d to L-GLU respectively, Table 18). Paired t-tests could s h o w no difference between either of these analogues a n d Q U I S either on the basis of antagonism by A P V or by KYNA. These c o m p o u n d s , like Q U I S , are significantly less sensitive to antagonism by A P V or by K Y N A than is N M D A (paired t-tests; Table 18).  Reduction of Amino Acid-Induced Activity of Dorsal Horn N e u r o n e s In vitro by Kynurenate  compound  averaae % reduction  X / N M D A (Daired)  NMDA  91.6 ± 6 . 2  (18)  D-cis-ACPD  92.5 ± 8 . 9  (6)  ( )1.01 ± 0.05 (6)  L-trans-ACPD  72.2 ± 18.4 (9)  ( a ) 0 . 9 4 ± 0 . 1 6 (9)  L-cjs_-ACPD  63.0 ± 13.6 (6)  (a)0.92 ± 0.10 (4)  QUIS  40.7 ± 16.9 (18)  (b)0.66 ± 0.29 (16)  QUIN  45.8 ± 2 3 . 1 (10)  ( ) 0 . 6 5 ± 0 . 1 7 (8)  2,5-PyrDA  47.0 ± 38.3 (7)  (b)0.55 ± 0.44 (7)  D-trans-ACPD  39.0 ± 25.6 (7)  ( )0.50 ± 0.52 (6)  L-GLU  30.3 ± 16.9 (9)  (b)0.48 ± 0.26 (6)  2,6-PyrDA  27.3 ± 23.9 (9)  (b)0.40 ± 0.36 (9)  a-not significantly different from NMDA. b -significantly different from NMDA.  1.00 a  b  b  paired t-tests p > 0.375. paired t-tests p < 0.05.  Table 20. Reduction of amino acid-induced firing of dorsal horn neurones in vitro by K Y N A expressed as the % reduction of control firing ± S.D. and as the X / N M D A ratio as described for Table 2 (page 52). The numbers of cells tested are given in brackets.  91  T h e s e d a t a are shown as the ranked order of paired results in Table 2 1 . W h e n m e m b e r s of this group of c o m p o u n d s (QUIS, 2,5- a n d 2,6-PyrDA) were c o m p a r e d with one another against A P V and K Y N A (top left quadrant of Table 21 for A P V a n d K Y N A ) , no distinction could be shown for 7 of 12 and 6 of 10 trials respectively.  In contrast, w h e n c o m p a r e d directly with N M D A against A P V and  K Y N A , NMDA-activity w a s more potently reduced by these antagonists than were m e m b e r s of this group in 35 of 35 and 24 of 28 trials respectively (top right quadrant of Table 21 for APV and KYNA).  L. Quinolinate - like activity Table 18 (page 85) shows that cell firing elicited by D-trans-ACPD w a s intermediately affected by A P V and w a s similar to Q U I N - i n d u c e d firing in its sensitivity to blockade by this antagonist (it w a s more susceptible than QUIS but less so than N M D A ) . Furthermore, as previously described, Figure 14, cell B. (page 84), demonstrates that D-trans-ACPD can retain considerable activity in the presence of an iontophoretic dose of KYNA which strongly attenuates N M D A and the NMDA-like isomers of A C P D . D-trans-ACPD w a s indistinguishable from Q U I N both on the basis of sensitivity to blockade by A P V and KYNA, a n d iontophoretic potency; both of these c o m p o u n d s require approximately 4 times the current of L-GLU to achieve similar firing rates from a given neurone (Table 18: page 85). In Table 19 (page 87) the ranked order of paired results shows that where D-lrans_-ACPD w a s c o m p a r e d directly with QUIN no distinction could be made by  92  Ranked Order of Paired Data for the Reduction of A m i n o Acid-Induced Activity of Dorsal Horn Neurones In vitro by D-(-)-2-amino-5-phosphonovalerate a n d Kynurenate  APV QUIS QUIS  0(3)  2.5- PyrDA  1 (3)  2.6- PyrDA  1 (4)  NMDA  0(0)  KYNA  2.5-PvrDA  QUIS  QUIS  2.6-PvrDA 3(4)  NMDA 16(0) 8(0) 11 (0)  0(0) 2.5-PvrDA 2(2)  2,5-PyrDA  1 (2)  2,6-PyrDA  0(4)  1 (0)  NMDA  0(2)  0(1)  Table 2 1 .  0(0) 2.6-PvrDA  NMDA  1 (4)  11 (2)  0(0)  5(1) 8(1)  0(1)  Antagonism of the amino acid-induced excitation of dorsal horn neurones in vitro by A P V and KYNA. Conventions are as described for Table 3 (page 53). The excitants are listed from left to right, and from above downward in order of increasing susceptibility to blockade. A bar indicates that the two compounds were never directly compared.  93  2,4-PyrDA 206  284  spikes/sec. 75  n  30nA 194  148  284  191  A  i  0 -I  I  •  15 s.  o o  I  o  QUIS 3 nA APV 292  242  • •  1  •  o N M D A 22 nA  5 nA  165  h  75  I  252  346  A  0 J  i  15 s.  •  1  o Q  1  r  15 s.  o  •  QUIS 5 nA  I  •  1  I  o  1  n  N M D A 25 n A  Figure 17. S e g m e n t s of a continuous ratemeter record of the responses of a single dorsal horn neurone in vitro to Q U I S (3 & 5 nA) a n d N M D A (22 & 25 nA) and the antagonism of those responses by: 2,4-PyrDA applied iontophoretically at 30 nA a n d A P V applied iontophoretically at 30 nA. Conventions as in Figure 9 (page 50).  94  A P V (2 of 3 cells) or K Y N A (2 of 2 cells); and it w a s clearly distinguishable from N M D A a n d the other isomers of A C P D (Table 19: page 87). Picolinate, 3-hydroxypicolinate, 2,4- and 3,4-PyrDA, phthalate a n d itaconate were also tested for activity in the spinal cord in vitro. These c o m p o u n d s were not able to excite dorsal horn neurones w h e n ejected from 200 m M solutions with currents up to 100 nA. The pyridine analogue 2,4-PyrDA w a s , however, found to reduce neuronal firing elicited by Q U I S , Q U I N , KAIN and N M D A .  Figure 17 shows  that 2,4-PyrDA applied at 30 nA from a 200 m M solution c a u s e d 25 a n d 32 % reductions of the firing elicited by Q U I S and N M D A respectively. A later trial with the s a m e cell s h o w e d that A P V applied at 5 nA completely blocked the firing induced by N M D A , while that by QUIS w a s 33 % lower than control.  2,4-PyrDA  w a s found to be very weak, however, requiring at least 200 times the relative iontophoretic dose of K Y N A to cause similar reductions of amino acid induced firing.  95  V.  Discussion  A. Interpretation of data and results All of the results to be discussed were obtained by extracellular recording of the electrical responses of dorsal horn and cortical neurones to iontophoretically applied amino acid excitants. The techniques used for iontophoresis a n d extracellular recording have some inherent features which should be noted prior to consideration of the results. For each experiment, the cell to be recorded w a s located by lowering the 7-barrelled electrode assembly through the tissue while ejecting an agonist, typically Q U I S or L-GLU. The agonist w a s often ejected continuously for several minutes such that many cells in the a r e a were affected. The oscilloscope signal s h o w e d only those cells nearest the electrode, with the closest of those producing the largest signal. Because this process discriminated against smaller neurones, the total population sampled w a s biased in favour of the larger cells in the area under study. This p h e n o m e n o n was aggravated by the use of a w i n d o w discriminator / ratemeter which permitted the experimenter to easily isolate large spikes, but made the selection of smaller signals more difficult.  If the cells in the  dorsal horn and cortex consist of two or more populations which differ in average size and in their responses to the amino acid excitants, then this biased selection of cells could represent a real problem. There is very little evidence, however, that populations of dorsal horn (ventral to the substantia gelatinosa) or cortical neurones vary qualitatively in their responses to excitatory amino acids.  96  Schneider  a n d Perl (1985) have reported that some superficial dorsal horn neurones do not respond to L-GLU, but no other supporting reports have been made.  Furthermore,  the author never observed neuronal responses to amino acid agonists or antagonists which w o u l d favour the supposition that the s a m p l e d cells represented heterogeneous populations which differed in their responses to the c o m p o u n d s e x a m i n e d (ie. the relative potency of agonists and antagonists w a s quite similar from neurone to neurone). Neurones which were particularly sensitive to excitation by one agonist invariably had higher than average sensitivities to all the agonists. T h e small variations in relative potency that were observed are likely due to the individual differences in current passing characteristics of each iontophoretic barrel and to the small differences in barrel-tip-to-cell distance for each of the six outer barrels of an electrode. Extracellular recording from neurones in the dorsal horn and cortex also involve other uncertainties regarding the positioning of the recording electrode, and thus the location of the c o m p o u n d source (iontophoretic barrel) relative to the s o m a , dendrites and axon hillock of the cell under study. It is the opinion of the author that c h a n g e s in the electrode to cell distance, and in the location of the electrode relative to the regions of the neurone mentioned above, c h a n g e d the apparent potencies of the c o m p o u n d s being ejected (a cell located a short distance from the ejecting barrel required a smaller iontophoretic dose of c o m p o u n d ) but did not alter the qualitative observations made from that cell. The variations in the responses between cells consisted entirely of small differences in the relative iontophoretic doses of agonists and antagonists needed to achieve the desired  97  effects. Cell to cell variations should not, and did not, result in observable qualitative differences in the excitation of the cells, or in the blockade of induced firing with antagonists. The experiments using antagonists applied in the superfusate were subject to small variations resulting from differences in the electrode to cell distance and the orientation of the electrode to the cell body: ie. a large cell s o m e distance from the electrode will be excited by having a comparatively large a r e a of m e m b r a n e e x p o s e d to a low concentration of agonist which is easily blocked by antagonist in the perfusate. By contrast, a cell located a shorter distance from the electrode may be excited by having a comparatively small a r e a of m e m b r a n e e x p o s e d to a high concentration of agonist which therefore requires s o m e w h a t higher doses of antagonist relative to the earlier scenario. W h e n care w a s taken to achieve stable, sub-maximal responses to the excitants, however, the blockade of the amino acid-evoked activity w a s also stable and consistent. The results showing the sensitivity of an agonist to blockade by an antagonist have been presented in three w a y s : the average % reduction of control firing pooled for all cells tested, the average % reduction of control firing c o m p a r e d directly with N M D A (X/NMDA ratio, paired), and the ranked agonists for each cell tested using a ± 15 % distinction value (every agonist is paired with every other on a small number of cells). The pooled data was subject to variations due to the differences in the electrode to cell distances and relative cell position and did not take those variations into account. The paired (X/NMDA ) results were obtained primarily from experiments where sub-maximal blockade of agonist-elicited activity  98  w a s achieved.  Because the reduction by an antagonist of the response of each  agonist w a s c o m p a r e d directly to the reduction of N M D A for each cell, the experimental conditions were virtually identical for each agonist, a n d the variabilities due to electrode position and cell location were reduced. Similarly, for the ranked paired d a t a , each number w a s generated by a direct c o m p a r i s o n of two agonists under similar experimental conditions, thus removing the variations e n c o u n t e r e d while using extracellular recording a n d iontophoretic techniques. A s s e s s m e n t of agonist or antagonist potency using iontophoretic currents as the measure is admittedly problematic. Table 11 (page 70) presents the average currents used during many experiments in vivo a n d in vitro to provide insight into a n d a possible explanation for the observation that Q U I N is capable of eliciting activity in vitro despite its lack of potency in the spinal cord in vivo.  Comparing  potencies in this w a y could only be done, however, because the numbers of cells involved were high, a n d the electrodes, solutions, and iontophoretic and recording conditions were similar. All the d a t a shown in Table 11 were obtained using the s a m e experimental techniques, electrodes and c o m p o u n d s from the s a m e suppliers, a n d , in many instances, agonist and antagonist dilutions derived from the s a m e stock solutions. The differences in currents used, therefore, probably represented a real increase in potency in vitro c o m p a r e d to in vivo, without an observable alteration to the qualitative results obtained with the various c o m p o u n d s (Table 11). Furthermore, no increase in the numbers of spontaneously active neurones w a s observed in vitro c o m p a r e d to the situation in vivo.  Reduced  uptake of c o m p o u n d s , possibly due to destruction of glia during the preparation of  99  the slices, may have contributed to, but is unlikely to have accounted entirely for the e n h a n c e d potency since no uptake systems are known for several of the compounds.  However, the increase in QUIN's potency (based on only a very few  cells in vivo) a p p e a r e d to be relatively greater than for the other agonists, and an observation made by the author that the potency of L-GLU w a s at least 3-fold higher (it w a s normally used at 500 m M in vivo but only 100 m M in 75 m M NaCI in vitro) w o u l d suggest that uptake efficiency for L-GLU a n d perhaps any degradative system available for Q U I N w a s reduced in the slice c o m p a r e d to the situation in vivo. W h y are Q U I S , N M D A , K Y N A and PDA more potent in vitro? A possible suggestion is that the replacement of the normal extracellular fluid with artificial cerebrospinal fluid (ACSF) may, because it lacks amino acids and protein, enhance the diffusion of ions from the electrode to the cell, thereby increasing the effective concentration reached for a given electrode-to-cell distance and iontophoretic current applied. Other unknown factors may, admittedly, have something to do with this observation. Determination of the relative potencies of agonists, as presented in Table 18 (page 85), presents less of a problem because much of the d a t a needed were obtained from paired comparisons. Nonetheless, the results achieved must be regarded as rough estimates and should only be interpreted as such.  More  accurate estimates of the potencies of these and other agonists in the spinal cord will be possible in the future using intracellular recordings from cells w h e r e the rate of rise and extent of depolarization of a cell in response to a topically or  100  iontophoretically applied agonist can be o b s e r v e d .  B. Excitation of dorsal horn neurones bv the archetypal agonists Q U I S . KAIN and N M D A in vivo and in vitro That the extracellularly recorded responses of dorsal horn neurones to the archetypal amino acid agonists Q U I S , KAIN and N M D A are mediated by three distinct receptors can only be inferred using the circumstantial evidence of their differential sensitivity to blockade by antagonists.  Unlike intracellular recordings  from CA1 neurones in the hippocampus (Peet et al., 1986) and pyramidal neurones in the cortex (Flatman et al., 1983) where these agonists elicit qualitatively different types of depolarization and spike activation, no such definitive distinctions can be made while recording extracellularly.  However, the responses  of dorsal horn neurones to these three agonists did have certain characteristics that were fairly consistent from cell to cell. As in the hippocampus (Peet et al., 1986), Q U I S - i n d u c e d excitations had a fast onset (indicative of a fast-rising depolarization) and had the most stable firing pattern of the three excitants, ie. firing reached its m a x i m u m rate quickly and stabilized at that rate, often giving a ratemeter peak with a distinct plateau. Increasing the iontophoretic dose of Q U I S s o m e t i m e s led to overdepolarization (indicated by a reduced extracellular spike amplitude), but required a relatively greater increase in the dose than for KAIN or NMDA. T h e responses to KAIN were unstable in comparison to those of Q U I S ; it was more difficult to find an iontophoretic dose which gave consistent sub-maximal  101  responses because small alterations in current could result in large c h a n g e s in the integrated firing rate. In addition, too large a dose of KAIN easily resulted in overdepolarization a n d occasionally c a u s e d apparently irreversible d a m a g e to the cell. T h e s e observations are consistent with those seen in the h i p p o c a m p u s (Peet et al., 1986), in particular the difficulty in achieving stable responses. The firing rate seldom attained a plateau, but rather t e n d e d to increase for the entire duration of agonist application. Both onset and offset of the responses were slower than for the other agonists, and continued firing after termination of the KAIN ejecting current w a s particularly noticeable. For these reasons fewer cells were recorded with KAIN than for the other agonists, and w h e n other analogues being examined were not KAIN-like in action it w a s not routinely tested (see for example Table 18: page 85). Bursts of action potentials induced by N M D A c o m m o n l y seen in other regions (Flatman et al., 1983; Peet et al., 1986) were never observed in the spinal cord. However, like KAIN, NMDA-elicited firing t e n d e d to increase in rate throughout the ejection period, although the offset was more rapid for N M D A . Toxic effects were seldom seen with high doses of N M D A , but overdepolarization occurred often with only moderate ( 1 5 - 2 0 %) increases in the iontophoretic dose, once a substantial but sub-maximal response had been obtained. In general, these observations are similar to those seen in the cortex and hippocampus (Flatman et al., 1983; Peet et al., 1986) despite the lack of NMDA-induced bursting in the spinal cord.  102  C. A n t a g o n i s m of dorsal horn neurone responses to archetypal agonists bv PDA. KYNA. A C R A and A P V in vivo To distinguish with confidence between the agonists acting at the different a m i n o acid receptors in the spinal cord using extracellular recording requires the use of at least two of the available amino acid antagonists. Experiments in the h i p p o c a m p u s (Peet et al., 1986) and cortex (Flatman et al., 1983) have shown that all c o m p o u n d s which elicit NMDA-like bursting and thus presumably activate N M D A receptors, are very sensitive to blockade by A P V , K Y N A a n d PDA. In the spinal cord it is p r e s u m e d that responses to a c o m p o u n d which are similar to those of N M D A in their sensitivity to blockade by these antagonists also act at N M D A receptors. In Table 2 (page 52), the antagonism of agonist-induced firing by PDA and K Y N A is s h o w n . PDA w a s first demonstrated to be an antagonist of amino acid excitations by Davies et al. (1981) in the cat spinal cord, w h e r e they found it to block N M D A excitations potently with lesser actions against the excitations produced by Q U I S a n d KAIN.  Their results were presented as pooled d a t a  showing N M D A responses to be reduced by 69 % with those of QUIS and KAIN by 54 and 55 % respectively. These data do not show a statistically significant difference between the agonists but nevertheless show trends similar to those presented in Table 2 (page 52). Other authors have reported that KAIN-induced excitations were similar in sensitivity to blockade by PDA as those of N M D A (McLennan a n d Liu, 1982). The order of agonist sensitivity to antagonism by PDA (the profile of antagonism) is similar to that of APV in the spinal cord, except the separation between agonists which can be achieved with PDA is less.  103  Cell firing e v o k e d by both N M D A and KAIN w a s powerfully attenuated by K Y N A , as s h o w n in Table 2 (page 52); QUIS-elicited activity w a s also affected, but to a lesser degree. T h e s e findings corroborate those of Peet et al. (1986) in the h i p p o c a m p u s , but differ s o m e w h a t from several other reports. For example, Perkins a n d Stone (1982, 1984) found that K Y N A had little ability to distinguish b e t w e e n N M D A a n d Q U I S excitations of either cortical or spinal cord neurones, although in the hippocampus they found that N M D A and KAIN were both more sensitive to K Y N A than w a s Q U I S . G a n o n g et al. (1983) reported that K Y N A was able to distinguish the activity of N M D A from that of Q U I N and Q U I S , and that the effects of KAIN were intermediately affected (more so than Q U I S but less than N M D A ) both in the hippocampus and immature rat spinal cord. In conclusion, the use of PDA or A P V to block NMDA-excitations and K Y N A to reduce both N M D A - and KAIN-like activity provides the needed circumstantial evidence to infer that Q U I S , KAIN and N M D A react with three distinct receptor types in the rat spinal cord in vivo.  D. A n t a g o n i s m of cortical neurone responses to amino acid agonists bv KYNA and A C R A in vivo T h e inclusion of Q U I N in the studies performed in the cortex in vivo w a s in response to the increasing interest in e n d o g e n o u s tryptophan-related c o m p o u n d s (including KYNA) and their potential roles as modulators of C N S excitability (for review see Stone and Connick, 1985). Furthermore, it w a s not possible to examine the pharmacology of QUIN in the spinal cord in vivo because of its marked lack of  104  potency in this preparation (Table 1 1 : page 70; Perkins a n d Stone, 1983; M c L e n n a n , 1984). A s reported by Perkins and Stone (1982, 1984) and M c L e n n a n (1984), Q U I N a n d N M D A - i n d u c e d excitations were affected almost equally by K Y N A in the cortex in vivo. In addition, the action of K Y N A against KAIN activity w a s also very similar to its antagonism of N M D A - i n d u c e d firing (Tables 4 and 5: pages 59 a n d 60). Only Q U I S w a s slightly less affected by KYNA in this preparation. T h e results presented here, in conformity with those of Perkins a n d Stone (1982, 1984) a n d M c L e n n a n (1984), provide evidence for the supposition that Q U I N exerts it excitatory action via N M D A receptors in the cortex as it does in the hippocampus (Peet et al., 1986). Because of the relatively poor separation between the excitations of the agonists provided by PDA and K Y N A in the cortex, any further conclusions would be difficult to d e f e n d . A C R A (Curry et al., 1986) had a non-specific antagonistic affect against all four of the agonists tested in the cortex, and provided no additional information regarding the receptors utilized by the various amino acid excitants.  E. The aromatic antagonists A C R A a n d K Y N A in the spinal cord and cortex The antagonistic actions of A C R A and KYNA provide important structureactivity information regarding the requirements for antagonism of amino acid excitations. Removal of the benzene ring from A C R A (Fig. 3, E: page 12) yielded Q U I N (Fig. 3, A), an excitant, even though the relative positions of the amino and carboxyl groups are unchanged. K Y N A devoid of its second aromatic ring is  105  4-hydroxypicolinate ^ - h y d r o x y s - p y r i d i n e carboxylate) which w a s not tested, but 2,4-PyrDA which w a s examined w a s found to be a rather w e a k antagonist in vitro (Fig. 17: page 94). It may therefore be concluded that an aromatic agonist is converted into an antagonist either by the addition of a second aromatic ring and/or by a change in position of the second acidic substituent from C3 to C 4 . Presumably the antagonistic effects of A C R A , K Y N A a n d 2,4-PyrDA are due to their ability to bind to but inability to activate the receptors because of steric factors (second ring) or the inappropriate placement of the distal carboxyl (C3 to C4) group.  F. a-Substituted analogues of glutamate In 1963, Curtis and Watkins reported that N-substitution of aspartate and glutamate (methyl, ethyl, propyl, iminomethyl) did not significantly reduce the potency of these c o m p o u n d s , and in some c a s e s dramatically e n h a n c e d it. It w a s considered possible that substitutions of this type made to the a-carbon rather than the nitrogen might enhance the binding of these c o m p o u n d s to receptors such that they w o u l d have potent actions either as agonists or antagonists. The analogues in this class that were synthesized and tested are listed in Table 1 (page 30). However, only the a-(parachlorophenyl)- a n d a - ( p a r a f l u o r o p h e n y l ) - s u b s t i t u t e d glutamates had any activity in the spinal cord in vivo ; they were w e a k antagonists with a preference for L-GLU-induced excitations (Tables 6 and 7: page 62). The iontophoretic potency of these c o m p o u n d s is low, but does increase w h e n going from the para-chloro to the para-fluoro analogue, indicating that steric interactions  106  with the aromatic ring are not necessarily the cause of the antagonistic actions. T h e increased tendency of the fluoride moiety (compared to chloride) to pull electrons off the ring and ultimately from the a-carbon suggests that the electronic effects of the substituted halogens may be the characteristic resulting in antagonism.  G. (+) and (-) Trans-1 -amino-1,2-cyclopentane dicarboxylate in vivo Examination of M t r a n s - C P A in the spinal cord shows that it is an antagonist with an action unlike any other c o m p o u n d previously reported save L-glutamate diethylester (GDEE). The iontophoretic potency of (-)trans-CPA is less than that of K Y N A , but greater than that of A C R A , and it is significantly more potent against Q U I S and KAIN activity than against that of N M D A (Table 8: page 66). The importance of this c o m p o u n d in the study of putative glutamatergic pathways in the spinal cord will take several years to determine, but its potential is great. Its action in the higher C N S , however, is entirely different from that in the spinal cord. Unpublished results obtained by intracellular recording from CA1 hippocampal neurones (M. J. Peet, personal communication) show (-)trans-CPA to be an agonist w h i c h elicits NMDA-like bursting and is blocked by APV. At no time did either isomer of t r a n s - C P A show excitant behaviour in the spinal cord. It is hoped that future studies with this c o m p o u n d will provide valuable information regarding the regional differences in the receptors for the excitatory amino acids. Figure 2, E and F (page 8) shows that there are two major differences in the structural characteristics of PDA and trans-CPA. First of all, PDA has a secondary  107  a m i n o group forming part of the piperidine ring, while the amino group of C P A is primary. Secondly, the relatively flexible piperidine ring of P D A may a s s u m e many of the conformations of cyclohexane rings, thus the intercarboxyl distances can vary considerably. The five-membered ring of CPA, on the other h a n d , is confined to an envelope conformation with relatively fixed inter-group distances (Table 25: page 130). The transition from an N M D A antagonist (PDA) to an antagonist with preference for Q U I S and KAIN-type excitations ((-)lrans-CPA) might be a c o n s e q u e n c e of the exclusion of the amino nitrogen from the ring a n d the reduction of the flexibility of the molecule which limits the c o m p o u n d to intercarboxyl a n d distal carboxyl to amino distances only attainable by L-GLU in a folded conformation. If trans-CPA binds to the Q U I S and/or KAIN receptors utilizing all three ionic groups, the spacing of the c h a r g e d areas on the receptor is limited by the distance over which electrostatic interactions may be effective. T h e QUIS-like actions of 2,5- a n d 2,6-PyrDA (discussed in more detail later) suggest that the distal carboxyl group is preferentially situated in juxtaposition to the amino group; ie. it is closer to the amino group than to the a-carboxyl. T r a n s - C P A should therefore be capable of binding with all three charged groups to a receptor with this configuration.  Nonetheless, trans-CPA binds with enough affinity to allow it to  c o m p e t e successfully with Q U I S , L-GLU and KAIN for these receptors. This supports suggestions that a partially folded conformation is the preferred one for the Q U I S (McLennan et al., 1982; McBain and Wheal,1984) and possibly for the KAIN receptor. That trans-CPA competes for QUIS a n d KAIN receptor sites but does not activate t h e m suggests that a folded conformation alone is insufficient for  108  activation of these receptors. Once bound to the receptor site flexibility in the agonist molecule permitting increases in the intercarboxyl distance may be important, perhaps allowing or encouraging a conformational c h a n g e in the receptor which is necessary for activation. T h e need for specific and potent QUIS and KAIN antagonists has not been filled, even by G D E E , which has in the past been used extensively as a Q U I S antagonist, but is being used less in recent years due to its s o m e w h a t unpredictable actions. More recent additions to the list of amino acid antagonists include several piperazine derivatives with potent N M D A - b l o c k i n g capabilities (Davies et al., 1986) a n d an analogue related to the dipeptide -yDGG, namely y-D-glutamyl-aminomethyl-sulphonate ( G A M S ) . G A M S has been used in the spinal cord in vitro (Davies and Watkins 1985) and in the thalamus (Salt, 1987) and w a s found in both regions to be a moderately potent antagonist with s o m e selectivity for responses to KAIN. None of the recently discovered antagonists have conformationally restricted structures and thus little structure-activity analysis c a n be done. It is apparent, however, by the number of these c o m p o u n d s containing co-phosphonate or sulphonate groups that this is a characteristic which confers potency to amino acid antagonists. T h e additional need for conformationally restricted c o m p o u n d s active at these receptors is now being met by cyclic amino acids such as those described here. The preliminary examination of trans-CPA discussed earlier suggests that it may be a useful tool to distinguish between synaptic events in the spinal cord which utilize Q U I S or KAIN receptors from those involving N M D A receptors.  109  Davies et al. (1982) reported on the actions of several piperidine derivatives, and concluded that the relative position of the carboxyl groups w a s the structural feature determining their effect.  The 2,3 arrangement of carboxyl groups provided  the most potent c o m p o u n d s in the series.  Jiaji§-2,3-piperidine dicarboxylate w a s  reported to be a potent N M D A agonist antagonized by A P V (Davies et al., 1982), while the cjs. isomer w a s , as also shown here (Tables 2 a n d 3: pages 52 and 53), an antagonist with a moderate preference for NMDA-excitations.  Davies et al.  (1982) also reported that PDA had a significant excitatory potency in the neonatal rat spinal cord in vitro, but not in the cords of frog or mature cat. Responses to this c o m p o u n d s h o w e d it to be more potent under these conditions than w a s L-GLU, and revealed that it w a s blocked by APV at iontophoretic currents which also blocked N M D A but had little effect on L-GLU. PDA was never found to be excitatory w h e n applied alone or concomitantly with another agonist to dorsal horn neurones either in vivo or in vitro, and as described above it had a significant potency as an antagonist against all amino acid-induced firing. That no excitatory properties were ever observed with PDA provides circumstantial evidence to suggest that the spinal cord slices prepared from 24 - 30 day old rats were mature with respect to the amino acid receptor populations. Davies et al. (1982) further reported that (-)cjs-PDA w a s responsible for both the agonistic and antagonistic actions of the racemic mixture. Also tested by Davies et al. (1982) w a s trans-2.4-piperidine dicarboxylate which proved to be a w e a k NMDA-like antagonist in both the rat and frog spinal cords. The flexibility of the piperidine ring, which can adopt many of the conformations of the cyclohexane ring, makes difficult  110  any structure-activity analysis beyond noting the importance of the 2,3- and 2,4placement of carboxyl groups for agonistic and antagonistic interactions with N M D A receptors respectively.  H. Archetypal agonists and blockade of their actions in the spinal cord in vitro T h e results of the initial experiments performed in vitro s h o w e d that the profile of antagonism (order of agonist sensitivity to blockade) for PDA, K Y N A and A P V w a s identical to that found in vivo. Furthermore, Table 10 (page 68) shows that the separation or distinction made between Q U I S / K A I N and N M D A by PDA and APV, and between Q U I S and K A I N / N M D A by K Y N A w a s greater in vitro than in vivo. This increase in selectivity allows the c o m p o u n d s to be divided into four statistically different g r o u p s (using paired Student's t-tests) on the basis of their susceptibility to antagonism (QUIS = L-GLU < KAIN < Q U I N < N M D A ; Table 14: page 76). These statistically significant differences show that Q U I S , KAIN and N M D A all act in a qualitatively different manner in the spinal cord, supporting on pharmacological grounds the theory that their actions are mediated by at least three different receptors.  Recent patch-clamp studies (Cull-Candy and Usowicz, 1987a, b) have  indicated that two or more binding sites as part of a single receptor-channel complex are responsible for the variety of responses observed to the amino acid excitants.  111  I. Actions of L- and D-glutamate in the spinal cord in vitro T h e investigations into the amino acid excitants in the early 1960's began by focussing on glutamate and aspartate, two c o m p o u n d s abundant in the C N S .  The  focus gradually shifted away from these e n d o g e n o u s excitants with the introduction of the more potent active c o m p o u n d s N M D A , a n d more recently, Q U I S and KAIN, which are c o m m o n l y u s e d today in the nomenclature of the receptors responsible for the actions of the excitant amino acids. It is still believed by most researchers, however, that the majority of amino acid mediated excitatory s y n a p s e s in the C N S use glutamate as the principal transmitter (see for review Puil, 1 9 8 1 ; F o n n u m , 1984). How this c o m p o u n d fits into contemporary ideas regarding the structural requirements for activation of the various receptor types should therefore be a d d r e s s e d . Tables 12 and 13 (pages 72 and 73) show the results of a series of experiments where L-GLU and D-GLU were c o m p a r e d directly with N M D A and Q U I S for sensitivity to blockade by APV and KYNA. Paired t-tests disclosed that L-GLU activity w a s indistinguishable from that of QUIS with respect to antagonism by A P V and KYNA, but that D-GLU w a s unlike either N M D A or Q U I S since it w a s affected by both A P V and KYNA (cf. Hicks et al., 1979). These results are most easily interpreted by suggesting that the structure of D-GLU allows it to activate either or both of the N M D A and KAIN types of receptor in addition to Q U I S receptors. The D-configured glutamate molecule appears to have a higher affinity for N M D A receptors and weakly activates those responding to Q U I S and KAIN. In the present experiments, L-GLU acted much more potently at QUIS receptors, although other studies have shown it to possess at least nominal affinity for the  112  N M D A or KAIN receptor varieties (for review see F o n n u m , 1984). The flexibility of the glutamate molecule allows it to fit any receptor template based on the configuration of one or more of the known potent excitants. It does not s e e m unbelievable to suppose that L-GLU may be responsible for mediating transmission at the bulk of "fast" excitatory synapses in the brain and spinal cord. T h e involvement of the different amino acid receptors in neuronal function w o u l d d e p e n d on their location, relative density, a n d balance of excitatory and inhibitory / antagonistic c o m p o u n d s in the extracellular fluid. The receptor type present at the highest concentration on the postsynaptic m e m b r a n e w o u l d determine w h e t h e r the postsynaptic response w o u l d be N M D A - or QUIS-like a n d therefore w h e t h e r or not C a  2 +  influx would a c c o m p a n y the N a influx during the +  E P S P . Many different roles for the archetypal amino acid receptors in synaptic activity have been d e t e r m i n e d ; a few, namely those in the hippocampus and spinal c o r d , were described in the introduction. E n d o g e n o u s ligands other than L-GLU a n d L-ASP have been discovered, a n d in particular the tryptophan metabolites Q U I N and K Y N A are strong candidates for roles as an e n d o g e n o u s agonist and antagonist respectively.  J. Does quinolinate act at a fourth receptor? There has been considerable discussion of the possible reasons for QUIN's relative lack of potency in the spinal cord and cerebellum in vivo (cf. Perkins and Stone,1983).  Perkins a n d Stone (1983) suggested that the observed differences in  potency support a role for QUIN as a transmitter since only small regional  113  differences in potency could be s h o w n for other excitatory amino acids, including L-GLU. These authors further suggested that these regional differences possibly indicate a heterogeneous population of N M D A receptors ( N M D A  1  and N M D A , 2  both A P V sensitive) which by way of differential distribution in the C N S may reflect a neurotransmitter function for Q U I N or a QUIN-like c o m p o u n d . Since there is no evidence for uptake of Q U I N anywhere in the C N S (see the review by Stone and Connick,.1985) or for a rapid enzymatic degradation of Q U I N (Foster et al., 1985), regional differences in such processes cannot readily be used to explain the differences in excitatory potency. T h e results to be discussed here (Tables 14 and 16: pages 76 and 81) show that w h e n using A P V to block the NMDA-elicited activity in the spinal cord in vitro, the excitation evoked by Q U I N was significantly less affected, but w a s more sensitive to blockade than those of Q U I S and KAIN. Iontophoretically applied APV thereby permits the agonists to be divided into three distinct groups, Q U I N being separated from the other agonists on the basis of its intermediate sensitivity to APV. The dose-response curves for A P V (Fig. 13: page 78) support this conclusion by showing a large separation between the effects of this antagonist applied topically on firing induced by N M D A , Q U I N , a n d Q U I S / K A I N . T h e IC50's determined from the dose-response curves for A P V in vitro show that to reduce the activities of N M D A , Q U I N and Q U I S by 5 0 % , concentrations of at least 2 x 10" , 7 x 1 0 6  6  and 1.8 x 1 0 " M would be required respectively (Table 15: 5  page 79). These d a t a are similar to those presented by ffrench-Mullen et al. (1986) for the piriform cortex in vitro, where N M D A , Q U I N and L-aspartate were c o m p a r e d  114  using the antagonist A P V (reported IC50's of 3.5 x 10" , 3.5 x 10" a n d 3 x 1 0 ' M 6  5  4  respectively). It is concluded, therefore, that on the basis of antagonism by APV, QUIN's action in the spinal cord in vitro is pharmacologically distinct from that of N M D A . This is in contrast to the actions of these c o m p o u n d s in the CA1 region of the h i p p o c a m p u s where Q U I N and N M D A are identical in their sensitivity to A P V (Peet et al., 1986, 1987) T h e actions of K Y N A in the spinal cord in vivo and in the h i p p o c a m p u s in vitro (Curry et al., 1986; Peet et al., 1987) are less specific than those of A P V (i.e. it antagonizes both KAIN and N M D A responses) and are about 10-fold less potent. In the spinal cord in vitro. KYNA applied iontophoretically (Tables 14 a n d 16: pages 76 a n d 81) w a s equally effective against KAIN and N M D A responses, a n d had a smaller action on responses elicited by Q U I S a n d Q U I N , which were similarly reduced (Tables 14 and 16). W h e n applied topically in a range of concentrations, K Y N A reduced the activities of N M D A and KAIN in parallel with significantly greater potency than it did those of Q U I N and Q U I S which were also reduced in parallel. The pattern of antagonism exhibited by K Y N A suggests that Q U I N does not excite spinal cord cells by acting, even in part, at either or both of the N M D A or KAIN receptors, since it w a s indistinguishable from QUIS using KYNA. That portion of QUIN's activity which w a s antagonized by A P V however, could not readily be accounted for by reaction with any of the known receptors since it w a s insensitive to KYNA, and thus it is suggested that a fourth amino acid receptor exists in the spinal cord, an inference which has also been made previously (Perkins and Stone, 1983).  115  A few experiments were performed using the spinal cord preparation in vitro with M g - f r e e m e d i u m . As described earlier, M g 2+  2 +  specifically blocks the action of  N M D A in a dose-dependent, voltage-sensitive manner (Evans et al., 1977; Davies and Watkins, 1977). Table 17 (page 82) shows that w h e n M g - f r e e medium was 2+  substituted for A C S F containing 2 m M M g , responses to both N M D A and QUIN 2 +  were approximately d o u b l e d while those of Q U I S were on average only 40 % larger. T h e inability of M g - f r e e bathing medium to distinguish between N M D A 2+  and Q U I N - i n d u c e d firing in the spinal cord suggests that these two receptors may be related, perhaps utilizing the same channel complex as apparently is the case in the hippocampus, in spite of their obvious pharmacological differences.  K. The actions of the isomers of A C P D and several pyridine derivatives related to quinolinate T w o dicarboxylate derivatives of pyridine (2,5- and 2,6-PyrDA), and all four isomers of A C P D (D- and L-, cjs.- and t r a n s - A C P D ) were found to be excitants in the spinal cord in vitro. Their actions were c o m p a r e d with those of L-GLU, Q U I S , Q U I N , KAIN and N M D A for susceptibility to blockade by A P V and KYNA.  Using  paired Student's t-tests, these agonists fall into three categories b a s e d on antagonism by A P V (Table 18: page 85); D-cis-. L-cis- and L-trans-ACPD are statistically indistinguishable from N M D A , D-trans-ACPD a n d Q U I N are similar to each other but statistically different from both N M D A a n d Q U I S , while 2,5- a n d 2,6-PyrDA are similar to L-GLU and Q U I S and statistically different from both N M D A and Q U I N . Using KYNA as an antagonist, the agonists divide into two  116  distinct categories, D-cis-. L-cis- and L-trans-ACPD being statistically similar to N M D A , while the remaining five all differ from N M D A but not from each other (Table 2 0 : page 91).  L. A n a l o g u e s acting at spinal cord N M D A receptors. T h e results of this study show that in the spinal cord, as in the hippocampus, D-cis-ACPD is a powerful excitant sensitive to both K Y N A a n d A P V . It is a structurally rigid D-amino acid with the two carboxyl groups in a cjs. configuration. Being the most potent conformationally restricted c o m p o u n d tested, it represents the best example of the preferred arrangement of charged groups for N M D A receptors in the spinal cord and hippocampus.  However, the responses of dorsal  horn neurones to L-cis- and L-trans-ACPD were also sensitive to A P V , in contrast to the hippocampus where they, a n d D-lrarjs.-ACPD, are insensitive to A P V but are blocked by K Y N A suggesting that they act via kainate receptors (Curry et al., 1987). It has been s h o w n here (Fig. 13: page 73) that KAIN-elicited firing of dorsal horn neurones w a s similar to QUIS-induced activity when A P V w a s used and w a s blocked in parallel with N M D A by K Y N A : none of the conformationally restricted c o m p o u n d s tested here s h o w e d this pattern of response. In the hippocampus the KAIN-like L-cis-. L-trans- and D-trans-ACPD are 4 to 8 times less potent than KAIN itself (Curry et al., 1987). In the spinal cord in vitro, however, indirect potency comparisons (relative to N M D A ) show t h e m be 35, 62 and 80 times less potent than KAIN respectively (L-trans-ACPD, L-gjs-ACPD and D-trans-ACPD: Table 18: page 85). The low potencies observed in the present  117  experiments c o m p a r e d to those found in the hippocampus suggest that these c o m p o u n d s may be unable to interact with KAIN receptors in the spinal c o r d , but w h e n applied at higher concentrations are able to activate N M D A receptors. Thus, it appears that differences may also exist in the structural requirements of the KAIN receptor a m o n g various regions of the C N S , a n d currently, a lack of other structurally related, conformationally restricted active c o m p o u n d s precludes a closer examination of this receptor type. It remains to be determined w h e t h e r or not the ability of L-trans- and L-cis-ACPD to activate the spinal cord N M D A receptor s h o w s an effect which is not observed in the hippocampus merely because these c o m p o u n d s act at hippocampal KAIN receptors at lower concentrations.  M. Quinolinate-like c o m p o u n d s A s mentioned previously Q U I N is pharmacologically indistinguishable from N M D A in the hippocampus (Peet et al., 1987) and cortex ( M c L e n n a n , 1984). By contrast, in the spinal cord it was relatively much less potent, w a s not readily blocked by K Y N A , and w a s not as sensitive to A P V as w a s N M D A (Fig. 13, Tables 18 and 2 0 : pages 78, 85 and 91). Phthalate (1,2-benzene dicarboxylate; Fig. 3, A: page 12), also NMDA-like in the hippocampus (Peet et al., 1987), is structurally similar to Q U I N but without the aromatic nitrogen, was unable to elicit firing of dorsal horn neurones in vitro even w h e n ejected at 70 to 100 nA from a 2 0 0 m M solution. In addition, phthalate neither e n h a n c e d nor d e p r e s s e d the activity of other agonists w h e n ejected concomitantly.  It is concluded that the aromatic  nitrogen of Q U I N is a requirement for activation of amino acid receptors by  118  heterocyclic agonists in the spinal cord. Furthermore, picolinate (2-pyridine carboxylate) and 3-hydroxypicolinate (Fig. 3, D and C: page 12), which are inactive in the h i p p o c a m p u s (Peet et al., 1987), were also unable to elicit activity in the spinal cord in vitro: however 3-hydroxypicolinate weakly e n h a n c e d activity elicited by other agonists indicating that it possesses a very slight excitatory ability. T h u s , it c a n be concluded that the carboxyl group on carbon 3 is important for the activity of heterocyclic agonists in the spinal cord, and substitution with an hydroxyl markedly reduces the ability of the c o m p o u n d to activate excitatory receptors. The failure of Q U I N to act at N M D A receptors in the spinal cord may be due to the fact that it has a shorter intercarboxyl distance than all the N M D A - m i m e t i c c o m p o u n d s tested.  D-tnms-ACPD (Fig.  14: page 84) is the other substance which,  possessing 1/4 the activity of L-GLU as an excitant in the c o r d , with respect to both potency and sensitivity to A P V and KYNA appears also to act at the same receptor as does Q U I N .  N. Quisqualate-like c o m p o u n d s In the absence of a specific L-GLU / QUIS antagonist, activation of this receptor is inferred from the relative insensitivity of evoked excitations to both APV and KYNA.  Both 2,6- and 2,5-PyrDA elicited firing in dorsal horn neurones in vitro  which w a s reduced by APV and KYNA to a degree comparable to Q U I S and L-GLU (Tables 18 and 20: pages 85 and 91). These c o m p o u n d s were found to possess about 1/5 the potency of L-GLU (Table 18); in contrast, QUIS w a s found to be 43 times more potent than L-GLU. The latter finding agrees with that reported by  119  several other authors (see e.g. Lodge et al., 1980). Despite the high iontophoretic d o s e s of 2,6- a n d 2,5-PyrDA required, it is unlikely that any activity w o u l d be o b s e r v e d in response to these pyridine derivatives if the c h a r g e d groups on the molecules were not appropriately positioned to interact with the receptor. 2,6-PyrDA w a s tested on cortical neurones by Perkins a n d Stone (1982) and was found to increase the spontaneous firing rate of a few cells but w a s extremely w e a k , a n d no report of the antagonism of the responses w a s given. A folded conformation allows the c h a r g e d groups of Q U I S and L-GLU to approximate a template based on 2,5- a n d 2,6-PyrDA (discussed in more detail in the following section). It is concluded, therefore, that the active conformations of Q U I S a n d L-GLU are those which approximate the conformations of the rigid 2,5a n d 2,6-PyrDA. This suggests that the y-carboxyl group (Cd) of L-GLU or the isoxazole ring of Q U I S is preferentially situated in juxtaposition to the a - a m i n o group rather than the cc-carboxyl (Cp) to interact with the receptor molecule.  The  comparative lack of potency of 2,5- and 2,6-PyrDA may be due to the presence of the aromatic ring and hindrance between it and c o m p o n e n t s of the receptor, or more likely that their arrangements of the charged groups are not optimal but nevertheless permit interaction with spinal cord Q U I S receptors, assuming some conformational flexibility in the latter. The significant drop in potency observed w h e n going from 2,6- to 2,5-PyrDA (t-test; p < 0.005) may thus reflect that the increased Cd - N distance in the latter c o m p o u n d places the distal carboxyl group in a less desireable position relative to the other charged groups than that seen in 2,6-PyrDA.  The lack of flexibility of these pyridine analogues could also be  120  responsible for their low potency.  If a conformational change in the receptor is  necessary for activation, this may be in part prevented or hindered by the rigidity of the 2,5- a n d 2,6-PyrDA molecules, thus reducing their effectiveness as activators once bound to the receptor. This supposition is also supported by the finding that (-)trans-CPA apparently competes for Q U I S receptor sites but is unable to cause excitation.  O. Regional differences in amino acid structure-activity relationships In 1968, M c L e n n a n and his colleagues reported that neurones in certain regions of the t h a l a m u s displayed significantly different relative sensitivities to L-GLU and DL-homocysteic acid. This paper w a s the first to provide evidence for the existence of heterogenous populations of receptors for the amino acids, and led the way to further reports strengthening the case in favour of transmitter roles for L-GLU a n d L-ASP.  There has since been an accumulation of evidence to  suggest that regional differences may exist not only in the distribution of the various receptors for the amino acids but also in their structure-function characteristics. Perkins and Stone (1983a) provided convincing d a t a showing that neurones in two central nervous system regions in particular did not respond as expected to NMDAmimetic agonists.  Dorsal horn and cerebellar neurones, w h e n recorded  extracellularly in vivo, were virtually insensitive to Q U I N despite its significant potency in the cortex and hippocampus. As already described, the increased potency of amino acid agonists in the spinal cord in vitro c o m p a r e d to in vivo enabled the pharmacology of Q U I N to be examined in this tissue. The d a t a already  121  discussed is in general similar to that presented by ffrench-Mullen et al. (1986) for the piriform cortex. An additional report by Herrling et al. (1983) places caudate neurones into the group typified by APV-sensitive bursts in response to both N M D A and Q U I N . It is tempting at this point, on the basis of the neuronal responses to N M D A a n d Q U I N , to divide the regions of the C N S into two groups: those in which N M D A a n d Q U I N are pharmacologically similar (cortex, hippocampus, striatum) and those in which Q U I N is relatively much less potent and is distinguishable from N M D A using A P V and/or K Y N A (spinal cord, cerebellum, piriform cortex). Care must be t a k e n , however, as pointed out by ffrench-Mullen et al. (1986), since although responses of piriform cortical neurones appeared to be very similar to those of spinal cord neurones with respect to many of the "standard" agonists a n d antagonists, w h e n examining G D E E it w a s found to have no effect on any of the amino acid or synaptic responses in the piriform cortex despite its well known effects in the spinal cord and elsewhere. However, this observation may have been due to the methods used ie. exclusively applying the antagonists in the superfusate and only at concentrations from 1 0 ' to 1 0 , since in the experience of 7  - 3  the author G D E E is not extremely potent w h e n superfusate applied to spinal cord neurones in vitro. Nevertheless, G D E E ' s lack of activity in the piriform cortex provides a warning against categorizing the regions of the C N S into discrete groups on the basis of response to the amino acids.  122  VI. Structure-activity relationships, model analysis  A. NMDA-like c o m p o u n d s . Figure 18 s h o w s , diagramatically, a template based on D-cis-ACPD which represents an arrangement of c h a r g e d groups with which NMDA-like c o m p o u n d s can react with in the spinal cord. Analysis of scale models of each of the a n a l o g u e s , using the bond length approximations s h o w n in Table 22 , has shown that the distance between the carboxyl carbon atoms of N M D A can vary between 0.25 a n d 0.37 nm (Table 23). The 5 - m e m b e r e d cyclopentane ring can adopt an envelope conformation where one carbon will extend out of the plane of the ring. For the c i s - A C P D isomers, the flexibility of the ring allows the distance between the proximal carboxyl (Cp; corresponding to the a-carboxyl of glutamate) a n d the distal carboxyl (Cd; corresponding to the y-carboxyl of glutamate) to vary between 0.33 a n d 0.45 nm (Table 23). In contrast, the distance between the carboxyl groups (Cd - Cp) of a model of L-trans-ACPD can vary only between 0.43 a n d 0.45 nm, while the C d - N distance has a range of 0.33 to 0.45 nm (Table 23). T h e s e c o m p o u n d s (L-cis- and L-trans-ACPD) were ca. 3 and 9 times less potent than N M D A (Table 18: page 85) and any receptor template based on these c o m p o u n d s should be d e s i g n e d taking into account their relative potencies.  The relative  positions of the charged groups of N M D A , D-cis- and L-trans-ACPD c o r r e s p o n d most closely if carbon 2 of D-cis-ACPD is out of the plane away from the amino group, allowing the separation of the carboxyl groups to approach its m a x i m u m of 0.45 nm (Table 23), while carbon 2 of L-trans-ACPD must also be out of the plane  123  of the ring but towards the amino group thus bringing the distal carboxyl to a position almost equidistant from the other charged groups. In this conformation, the distance separating the distal carboxyls from the amino groups of both A C P D isomers is about 0.44 n m . N M D A can adopt a conformation with an intercarboxyl distance of 0.35 nm (Table 2 3 ) ; in this conformation C d is also virtually equidistant from both N a n d C p a n d is approximately 0.09 nm closer to the proximal groups than in the D-cjs-ACPD model.  Bond Lengths Used With the Orbit Molecular Building S y s t e m bond  interionic distance  - C-C-  0.15 nm  - C= C-  0.14 nm  -C-N-  0.14 nm  -C = N-  0.13 nm  T a b l e 22. Bond length estimates used in the construction of chemical models for structure-activity analysis. Model system used is the Orbit molecular building system, Cochranes of Oxford.  L-cis-ACPD can fit precisely the template based on D-cis-ACPD (Fig. 18: page 133). It has a similar arrangement of charged groups, but has ring carbons extending into the plane of the presumed active site (L-cis carbons 4 a n d 5). The protrusion of the rings of this analogue into the binding region may sterically hinder its approach to the receptor site and be responsible for its lower potency c o m p a r e d to D-cis-ACPD. L-cis-ACPD w a s 16 times while D-cis-ACPD w a s 3 times less potent than N M D A (Table 18: page 85).  124  Interionic Distances Determined by Analysis of Molecular Scale Models  A. Compound  D-cis-ACPD * range  L-cJ£-ACPD * range  L-trans-ACPD * range  NMDA* range  QUIN D-trans-ACPD * range  B.  C d - Cp (nm)  0.44  0.45 0.33 - 0.45  0.43 - 0.45  0.44  0.45 0.33 - 0.45  0.43 - 0.45  0.44  0.45  0.43 - 0.45  0.33 - 0.45  0.33  0.35 0.25 - 0.37  0.25 - 0.37  0.27  0.36  0.44  0.45  0.43 - 0.46  0.33 - 0.46  B. / A. 0.98 0.98 - 1.30  0.98 0.98 - 1.30  1.02 0.77 - 1.02  0.94 0.71 - 1.40  1.33 1.02 0.77 - 1.02  2,6-PyrDA  0.47  0.23  0.49  2,5-PyrDA  0.55  0.36  0.65  QUIS * range  L-GLU * range  0.22  0.38 0.18 - 0.50  0.18 - 0.50  0.38  0.22  0.16 - 0.50  0.16 - 0.50  Table 23. *  Cd - N (nm)  0.58 0.48 - 2.06  0.58 0.48 - 2.06  Interionic distances estimated from molecular scale models constructed using the "Orbit Molecular Building System". Compounds in italics are those from which templates have been constructed for Figure 18 (page 133). bond lengths determined with molecule in conformation of best fit for templates in Figure 18. A C2-envelope conformation for A C P D analogues, an extended conformation for N M D A and a folded conformation for QUIS and L-GLU.  125  B. QUIN-like c o m p o u n d s . Unlike the cases of the NMDA-like isomers of A C P D discussed above however, it is difficult to construct a simple template model which will accept both QUIN and D-lnans-ACPD. The intercarboxyl distance C d - Cp, and the C d - N and Cp - N distances are fixed in Q U I N at 0.27, 0.36 and 0.23 nm respectively, while the corresponding distances for D-trans-ACPD lie in the ranges 0.43 - 0.46 and 0.30 - 0.46 with the Cp - N length fixed at 0.24 nm (cf. Table 23), a n d there is no evident w a y in which the two molecules can be matched to a single template. W h e t h e r this betokens the existence of yet another receptor subtype, or whether the postulated Q U I N receptor is sufficiently malleable w h e n confronted with an unusual agonist that its configuration can change to accept a molecule with a quite different charge separation, is unclear. Neither alternative is particularly attractive.  C. QUIS-like c o m p o u n d s . Analysis of scale models of L-GLU and Q U I S demonstrates that the distances b e t w e e n the distal carboxyl and the amino groups (Cd - N) of the rigid pyridine analogues are 0.23 and 0.36 nm for 2,6- and 2,5-PyrDA respectively (Table 23). A template has been constructed based on these c o m p o u n d s (Fig. 18: page 85) a n d this has been c o m p a r e d to models of Q U I S and L-GLU. Assuming that the isoxazole ring of QUIS plays the same role in binding as does the distal (y) carboxyl group of L-GLU, the Cd - Cp and C d - N distances of these c o m p o u n d s are shown in Table 23. A folded conformation allows the c h a r g e d groups of QUIS and L-GLU to approximate a template based on 2,5- and 2,6-PyrDA, with a Cd - N  126  distance close to that of the latter but 0.14 nm shorter than the former c o m p o u n d . T h e s e results place 2,5- and 2,6-PyrDA on the end of a short list of c o m p o u n d s k n o w n to interact with spinal cord Q U I S receptors. Also included are L-GLU, L-cysteate, and a n u m b e r of c o m p o u n d s structurally related to ibotenic acid, including (±)-a-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid ( A M P A ; Krogsgaard-Larsen et al., 1980; Krogsgaard-Larsen, et al., 1981). All of these c o m p o u n d s are structurally capable of achieving a conformation where the distal anionic group is in juxtaposition with the amino group, despite the conformational restrictions present in those related to A M P A . The structural feature c o m m o n to all the potent QUIS-like agonists, which is not observed in those c o m p o u n d s acting at the other amino acid receptors, is the ability of the c o m p o u n d to adopt a conformation where the distal carboxyl group (or isoxazole ring) is less than 0.24 nm distant from the amino group (ie. C d - N ca. 0.24 nm). This feature is seen in L-GLU, Q U I S and A M P A , three powerful excitants, and also in 2,6-PyrDA, all of which show similar profiles of sensitivity to antagonism (Krogsgaard-Larsen, 1980). Table 24 characterizes a series of analogues related to A M P A which have different potencies and sensitivities to antagonism in the cat spinal cord (Krogsgaard-Larsen et al., 1980; Lauridsen et al., 1985). A M P A - and homoibotenate-elicited activity is mediated via L-GLU / QUIS receptors as it is sensitive to blockade by G D E E while that of ibotenate is not. A M P A is approximately 3 times more potent than L-GLU while homoibotenate is slightly less; the activities of both are insensitive to blockade by the N M D A antagonist D a A A . The potency of ibotenate is twice that of L-GLU, but firing induced by this  127  c o m p o u n d is not blocked by G D E E but is susceptible to antagonism by D a A A . If the activity of these c o m p o u n d s is dependent on the intercarboxyl (Cd - Cp) distance, ibotenate should be more potent than homoibotenate (being closer to A M P A in this respect) as it is, but also should be GDEE-sensitive (QUIS-like), which it is not. A structure-activity parallel can be s e e n , however, for the potency a n d sensitivity to antagonism based on variation in the Cd - N distance, and/or in the ratio of C d - N (B) / Cd - C p (A) (Tables 23 and 24). A M P A has a m i n i m u m Cd - N of 0 . 2 1 , a B/A ratio of 0.54, and is L-GLU-like in its sensitivity to G D E E . The minimum C d - N distance seen in homoibotenate is 0.33 nm with a B/A ratio of 0.67, and it is described as being less potent than L-GLU but sensitive to G D E E . In contrast, ibotenate shows a minimum Cd - N distance of 0.40 n m , a B/A ratio of 0.91 and is not blocked by G D E E . Assuming that the isoxazole ring of homoibotenate has an a r e a of electrostatic interaction larger than that of the carboxyl group of L-GLU (an assumption supported by the 43 fold increase in potency w h e n going from L-GLU to Q U I S ) , homoibotenate can conceivably interact with a receptor having the c h a r g e d groups located ca. 0.24 nm apart. The antagonism of L-GLU a n d Q U I S activity by H t r a n s - C P A also supports these conjectures; it is limited to a Cd - N distance of 0.25 nm and has a B/A ratio of 0.69 (Table 25) a n d should be capable of interacting with the L-GLU / QUIS receptor template shown in Figure 18.  128  Interionic Distances of Other Conformationally Restricted A n a l o g u e s Determined by Analysis of Molecular Scale Models  A. Cd - C p (nm) compound cvclic a n a l o g u e s : AMPA  0.38  1  0.21 - 0.48  range  homoibotenate range  0.48  1  0.33 - 0.53  ibotenate range  0.44  1  0.40 - 0.44  A MA A  0.33  range  0.28 - 0.39  B. Cd - N (nm)  0.21  potency*  ++++++  G D E E * DaAA*  yes  no  0.21 - 0.48  0.33 0.33 - 0.53  0.40  0.54 0.54 - 1.85  +(+)  yes  ++++  no  no  0.67 0 . 6 7 - 1.50  yes  0.40 - 0.44  0.91 0.91 - 1.10  ++  0.33  B/A  no  yes  0.28 - 0.39  1.00 0.70 - 1.40  bicyclic a n a l o a u e s : 7-HPCA  2  0.48  0.38  ++++  yes  no  0.80  5-HPCA  2  0.50  0.40  ++++  yes  no  0.80  4-HPCA  2  0.35  0.40  n.a.  inactive  Table 24.  1.14  Interionic distances estimated from molecular scale models constructed using the "Orbit  Molecular Building System". * 1. 2.  Potencies (relative to L-GLU = ++) and sensitivities to G D E E and D a A A in the cat spinal cord in vivo from Krogsgaard-Larsen et al., 1980, 1985. bond lengths determined with molecule in conformation of best fit for the QUIS/L-GLU template in Figure 18 (page 133). bond lengths determined with molecule in conformation described by Krogsgaard-Larsen et al., 1985.  129  Interionic Distances of Conformationally Restricted Antagonists Determined by Analysis of Molecular Scale Models A. Cd - Cp (nm)  Compound Mtrans-CPA ' 1  range  PDA" " 1  range  Table 25. t *  0.36 0.33 - 0.36  0.28 0.25 - 0.28  B. C d - N fnm)  QUIS*  NMDA*  0.25  yes  no  0.69 0.69 - 0.76  0.24 - 0.26  0.28  B./A.  no  1.00  yes  0.28 - 0.37  1.00 - 1.36  Interionic distances estimated from molecular scale models constructed using the  "Orbit Molecular Building System", bond lengths determined with molecule in conformation of best fit for the QUIS / L-GLU template in Figure 18 (page 133). reduce excitations elicited by iontophoretic application of QUIS or N M D A  D. Other conformationally restricted c o m p o u n d s Many other conformationally restricted c o m p o u n d s related to ibotenate have been synthesized a n d tested by Krogsgaard-Larsen a n d his colleagues (1980, 1 9 8 1 , 1985), the actions of which provide additional support for the structureactivity distinctions between QUIS a n d N M D A receptors discussed previously. (RS)-3-hydroxy-4,5,6,7-tetrahydroisoxazolo[5,4-c]- pyridine-7-carboxylic acid a n d 5-carboxylic acid (7-HPCA, 5-HPCA; Krogsgaard-Larsen et al., 1985), (RS)-3hydroxy-4,5,6,7-tetrahydro- isoxazolo[5,4-c]pyridine-4-carboxylic acid (4-HPCA; M a d s e n et al., 1987) a n d a-amino-5-methyl-3-hydroxy-4-isoxazoleacetic  acid  ( A M A A ; Krogsgaard-Larsen et al., 1980; Honore et al., 1981) are four conformationally very restricted c o m p o u n d s ; 4 - H P C A is inactive and A M A A is NMDA-like, while 5- a n d 7-HPCA are G D E E -sensitive but are unaffected by D a A A and are therefore QUIS-like. T h e charged groups of A M A A are held on an aspartate-like backbone which is confined by the isoxazole ring a n d the methyl  130  g r o u p on carbon 5 into an extended conformation (Krogsgaard-Larsen et al., 1980) with a B/A ratio near 1.0 (Table 24), hence its NMDA-like activity. T h e charged groups of 4 - H P C A are separated by one additional carbon (thus with a glutamate b a c k b o n e ) , however the bicyclic nature of this c o m p o u n d constrains the receptive g r o u p s into a configuration with a B/A ratio of ca. 1.2 (Table 24) apparently rendering it unable to activate either N M D A or QUIS receptors. Of this group of c o m p o u n d s only 5- a n d 7-HPCA are configured such that the intercarboxyl distance (Cd - Cp) is greater than that separating Cd from N, and an interaction with Q U I S receptors is observed. T h e s e analogues have a B/A ratio of ca. 0.8 w h e n in the conformations described by Krogsgaard-Larsen et al., (1985), which is greater than that possible in other analogues e x a m i n e d , but still allows a sufficient degree of folding for interaction with Q U I S receptors. T h e s e c o m p o u n d s are unable to achieve a conformation where the B/A ratio is 1.0, thereby preventing their interaction with N M D A receptors. Neither 5- nor 7-HPCA has a potency comparable to Q U I S or A M P A however, supporting further the suggestions made earlier in this discussion that a conformation allowing the distal anionic group to c o m e into close juxtaposition with the amino group is preferred by Q U I S receptors and that structural rigidity may not be entirely conducive to potency at this receptor type. All of the agonists tested which are capable of achieving a B/A ratio less than 0.70 are found to be G D E E sensitive (Krogsgaard-Larsen et al., 1980) and/or are QUIS-like with respect to both KYNA a n d APV (Tables 23 and 24). Furthermore, those agonists confined to a conformation with a B/A ratio of 0.8 (5- and 7-HPCA)  131  have also been reported to be QUIS-like and presumably are unable to interact with N M D A receptors. In addition, the two conformationally restricted antagonists studied here, PDA and trans-CPA also fit this model, since PDA has a minimum B/A ratio of 1.00 a n d c o m p e t e s primarily with NMDA-like agonists while t r a n s - C P A , which has a m i n i m u m B/A ratio of 0.69, effectively blocks QUIS-like agonists (Table 25).  VII. Contributions made to the study of the spinal cord and excitatory amino acids in the central nervous system  In vitro slice preparations have been used successfully to study the function of the central nervous system for more than 20 years. For several reasons, the number of successful attempts to examine the adult m a m m a l i a n spinal cord as a slice, to date, is very small. First of all, the ease of preparation of slices of the cortex and h i p p o c a m p u s a n d their highly organized cytoarchitecture make t h e m desirable subjects of study. Secondly, the spinal cord has been studied successfully in vivo for many years, and the structure itself and its afferent and efferent innervation are anatomically well defined and accessible.  Finally, great difficulties are associated  with the preparation of spinal cord slices because of the high proportion of white matter present, and the softness of the tissue even when chilled. The evolution of the techniques available to prevent d a m a g e during the cutting process led to the successful development of this preparation. It is hoped that this method of spinal cord slice preparation, or a modification thereof, will be successfully adopted by other laboratories wishing to study the function of the spinal cord.  132  Q u i s q u a l a t e / L-glutamate (2,5- and 2,6-PyrDA)  0.40 - 0.45 n m 0.22 - 0.26 n m  0.24 n m  NMDA  (D-CJ2-ACPD)  0.35 - 0.45 n m  0.33 - 0.43 n m  0.24 n m  Figure 18. Diagrammatic representations of templates d e s i g n e d to a c c o m m o d a t e the ligands acting at Q U I S a n d N M D A spinal cord receptors. A s d r a w n , the QUIS receptor template a c c o m m o d a t e s a range of C d - N distances of 0.22 - 0.26 n m , and a B / A ratio of about 0.50 - 0.80. T h e N M D A receptor template has a B / A ratio near 1.00.  133  VIII. Structure-Activity Conclusions  It has been s h o w n that the requirements for activation of N M D A receptors in the spinal cord involve an adjacent positioning of two carboxyl g r o u p s a n d the presence of an amino group, either primary or secondary, located in a D-configuration with respect to the carboxyl groups. Aromatic agonists (QUIN and pthalate) are not accepted by spinal cord N M D A receptors as they are in the hippocampus.  Ring c a r b o n s extending into the region of the c h a r g e d binding  groups as in L-cis-ACPD appear to hinder the interaction of the agonist with the spinal cord receptor; and the addition of a second ring as in K Y N A results in an antagonist for both N M D A and KAIN.  All of the conformationally restricted  a n a l o g u e s tested which were found to be A P V sensitive are capable of achieving a conformation where the distal carboxyl group is approximately equidistant from the proximal c h a r g e d groups giving a B/A ratio close to 1.0 (Table 23: page 125). Two isomers of A C P D which are KAIN-like in the hippocampus are NMDA-like in the spinal cord suggesting that differences also exist between spinal cord a n d hippocampal KAIN receptors. Activation of the receptor(s) with which Q U I N interacts (the " Q U I N receptor") requires one cationic group and two rather widely s p a c e d anionic g r o u p s ( D - t r a n s - A C P D ) . a n d substitution (3-hydroxypicolinate) or removal (picolinate) of the 3-carboxyl group greatly reduces or eliminates activity. Substitution of a sixth aromatic carbon for the aromatic nitrogen of Q U I N (phthalate) also results in a loss of activity. Only QUIN and D-trans-ACPD are known to cause excitations in the  134  spinal cord which show this pattern of antagonism (QUIS-like in senstivity to KYNA, intermediately affected by A P V ) . This pattern of antagonist sensitivity has not been observed for any agonist in the hippocampus, but has in part been described in the piriform cortex (ffrench-Mullen et al., 1986). With respect to the orientation of the three binding groups these two c o m p o u n d s are structurally dissimilar, and it is impossible to draw any defensible structure-activity conclusions b e y o n d those m e n t i o n e d above. Spinal cord Q U I S receptors also require one cationic and two anionic groups, but prefer the distal anionic group to be adjacent to the cationic group as it is in 2,5and 2,6-PyrDA. This supports suggestions that QUIS and L-GLU interact with spinal cord Q U I S receptors in a folded conformation (McLennan et al., 1982). Decreases in the potencies of QUIS-like agonists c a n be observed w h e n the m i n i m u m Cd - N distance is greater than 0.24 n m , and/or w h e n the B/A ratio described in Table 23 (page 125) is greater than 0.60 (AMPA to homoibotenate; 2,6-PyrDA to 2,5-PyrDA). Shifts from agonism via QUIS receptors to that via N M D A receptors is observed when going from L- to D-configured excitants (L-GLU to D-GLU) and/or w h e n going from close analogues with B/A ratios increasing b e y o n d 0.70 (homoibotenate to ibotenate; L-GLU to N M D A ; Tables 23 and 24: pages 125 and 129). The relatively large discrepencies in the Cd - Cp and C d - N distances seen in c o m p o u n d s which apparently act at similar receptors in the spinal cord (L-trans-ACPD and D-cjs-ACPD; 2,5- and 2,6-PyrDA) suggests that the tolerance of the receptor binding sites may be quite large, particularly for the distal or y-carboxyl  135  moiety, allowing the relative positions of binding groups to vary by more than 0.10 nm in s o m e situations. Although electrostatic interactions are thought to occur over relatively long distances, it is unlikely that three different arrangements of receptive g r o u p s carrying "single" charges similar to those associated with carboxyl and a m i n o groups in solution at pH 7.4 could account for these observations. A greater degree of latitude could be imagined, however, if the c h a r g e d groups on the receptors were larger, possibly c o m p o s e d of two or more similarly c h a r g e d moieties, allowing the binding site to be considered a c h a r g e d region or area rather than an individual group. Alternatively, the receptive groups may be c o m p o s e d of amino acids with little conformational restriction allowing t h e m to adopt one of a number of different conformations in order to a c c o m m o d a t e ligands of various sizes, or various conformations of a particular ligand. This explanation is made even more plausible w h e n considering the large variety of acidic amino acids of varying chain length which are capable of interacting with these receptors. The similarity in the B/A ratios seen in those conformationally restricted c o m p o u n d s found here to act either at QUIS receptors (0.49 - 0.69) or N M D A receptors (0.77 - 1.02) supports the suggestion that the receptive group which interacts with the distal carboxyl is either mobile or large enough to a c c o m m o d a t e c o m p o u n d s with different distances between this moiety and the oc-carbon. Finally, there is evidence to suggest that more than one amino acid molecule is necessary to activate excitatory receptors, and thus some degree of cooperativity in the interaction of ligand(s) and receptor may exist (McLennan and W h e a l , 1976).  136  In conclusion, the use of conformationally restricted analogues of glutamate, aspartate and quinolinate have provided new information regarding the structural requirements for activation of spinal cord amino acid receptors. The evidence in support of a fourth amino acid receptor, responsible for the activity of quinolinate in the spinal cord, has been strenghtened and details of the regional differences in the a m i n o acid receptors have been accumulated. The fundamental differences in the N M D A receptors of the spinal cord and hippocampus a n d the possible involvement of the latter in learning and memory (via long-term potentiation) suggests that functional significance may, in the future, be attributed to the structural differences seen in these studies. Local patterns of distribution may also be an important correlate of function, as has been suggested for the cerebellum (Olsen et al., 1987).  137  IX. References Anis, N.A., Berry, S.C., Burton, N.R. a n d Lodge, D. (1983) The dissociative anesthetics, ketamine and phencyclidine, selectively reduce excitation of central m a m m a l i a n neurones by N-methyl-D-aspartate. Br. J. Pharmac., 8 3 : 1 7 9 - 185. Avoli, M. a n d Olivier, A. (1987) Bursting in human epileptogenic neocortex is d e p r e s s e d by an N-methyl-D-aspartate antagonist. Neurosci. Lett. 76: 249 - 254. Baudry, M. and Lynch, G. 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