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The interactions of putative neuroprotectant compounds with NMDA ion channels Sawyer, Dale C. 1997

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THE INTERACTIONS OF PUTATIVE NEUROPROTECTANT COMPOUNDS WITH NMDA ION CHANNELS  (  by DALE C.SAWYER B.Sc, University of British Columbia, 1987 M.Sc, University of British Columbia, 1992  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Pharmacology & Therapeutics, Faculty of Medicine)  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BlUTISlf COLUMBIA May 1997 ©Dale C. Sawyer, 1997  In  presenting  degree at the  this  thesis  in  University of  partial  fulfilment  of  of  department  this thesis for or  by  his  or  requirements  for  an advanced  British Columbia, I agree that the Library shall make it  freely available for reference and study. I further copying  the  agree that permission for extensive  scholarly purposes may be granted her  representatives.  It  is  by the  understood  that  head of copying  my or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department  of f  H Aft MA CD/ ;OGV ft lVl<?XAPmi  The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  Q'CTOfegft  ( g  \<\<\%  ABSTRACT The NMDA (/V-methyl-D-aspartate) subtype of the receptors for the excitatory amino acid L-glutamate has been implicated as a mediator of anoxic neuronal death following  periods of cerebrovascular ischaemia.  Laboratory observations have  implicated it in neuronal death occurring in other situations, including hypoglycemia and neurodegenerative disorders.  Inhibiting the NMDA receptor could, therefore,  represent a useful therapeutic intervention where C N S damage is a potential outcome resulting from conditions such as those given above. However, since NMDA receptors are involved in a number of vital CNS functions, such as synaptic transmission, simple all-or-none antagonism would present an unacceptable risk of toxicity and side effects. Determination of specific characteristics of a given agent's interaction with the NMDA receptor-ion channel complex would aid considerably in assessing that agent's clinical utility as a safe and efficacious neuroprotectant. The ion channel associated with the NMDA receptor is the target of a number of uncompetitive NMDA antagonists. Four such agents have been chosen for study:  (-)-  and (+)-p-cyclazocine are members of the benzomorphan class of compounds, several others of which are known to act as NMDA antagonists; dextromethorphan (DM) is an antitussive morphinan closely related in chemical structure to the benzomorphans; and L-687,384, which is, like a number of other uncompetitive NMDA antagonists, a ligand for sigma receptors. These agents were tested for their actions against responses to NMDA. As an estimation of the potential for producing side effects, (-)-p-cyclazocine and DM were also studied for their effects on responses to the non-NMDA glutamate receptor  agonists  kainate  and  a-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazole-  - Ill -  propanoic acid (AMPA), as well as for their actions against voltage-gated C a * , N a , 2  +  and K* currents. Finally, (-)-p-cyclazocine was tested in a cell viability assay to confirm its efficacy as a neuroprotective agent. Unitary currents through single NMDA-activated ion channels were studied using outside-out patches isolated from cultured rat hippocampal or cortical neurons. Currents recorded in the presence of NMDA were compared to those recorded following the addition of one of the agents. None of the compounds studied altered the amplitudes of the unitary currents activated by NMDA and hence did not affect the channel conductance.  The compound (-)-p-cyclazocine  concentration-dependently  reduced the open state probability (P ) of NMDA ion channels with 0  IC50  values of 84 nM  and 680 nM in hippocampal and cortical neurons, respectively. The reduction in P by 0  (-)-p-cyclazocine was attributable to decreases in mean channel .open time and mean channel opening frequency. In hippocampal neurons, (+)-p-cyclazocine was 170x less potent in reducing P than the (-)-enantiomer 0  (IC50  = 14 pM for (+)-p-cyclazocine).  DM  also reduced P , with IC50 values of 4.4 pM and 3.8 pM in hippocampal and cortical 0  neurons, respectively. Again, decreases in mean open time and mean frequency were associated with the reduction in P by DM. L-687,384 decreased the mean open time 0  of  NMDA-activated  unitary  currents,  but  did  not  diminish  Consequently, the action of L-687-384 against P was relatively weak 0  their (IC50  frequency. = 61 uM).  The reductions in mean open time and mean frequency by (-)-p-cyclazocine and DM, without effects on channel conductance, were consistent with an open-channel block model for inhibition of NMDA activity. Analysis of the results assuming an openchannel blockade model gave estimations of the on-rate constants (k ) and off-rate 2  - iv constants (k. ) for the interactions of the compounds with NMDA ion channels in 2  hippocampal and cortical neurons. The k values in both neuronal types were similar 2  for (-)-p-cyclazocine, DM, and L-687,384, all being near 10 M'V . 7  The closeness of  1  the k values suggests that the on-rate for a given open channel blocker is not a 2  determinant of its potency as an NMDA activity inhibitor. The frequency reductions by (-)-p-cyclazocine and DM allowed for estimations of k , an apparent unblocking, or 2  "off, rate constant, from the single-channel data; this was 2.5 - 5.0 s" and 2.5 s' for (-)1  1  p-cyclazocine in hippocampal and cortical neurons, respectively, and 10 - 13 s" for DM 1  in hippocampal neurons. Optical  recording  of  intracellular  free  calcium  [Ca ]j 2+  responses using  hippocampal or cortical neurons loaded with the Ca *-sensitive fluorescent dye fura-2 2  allowed further elucidation of the actions of (-)-p-cyclazocine and .DM on responses to NMDA. Both agents depressed NMDA responses with ICso values of 270 nM and 220 nM for (-)-p-cyclazocine in hippocampal and cortical neurons, respectively, and 4.1 uM and 5.4 uM for DM in hippocampal and cortical neurons, respectively. The action of (-)p-cyclazocine was use-dependent, a property consistent with open-channel block. No effect on NMDA-evoked  [Ca *]i 2  responses was observed with (+)-p-cyclazocine in either  neuronal type. In either hippocampal or cortical neurons loaded with fura-2, (-)-p-cyclazocine at 5 uM was found to have no action against [Ca *]i responses to the non-NMDA agonists 2  kainate and AMPA.  Responses evoked by exposure of the neurons to high [K*]-  containing medium were also unaffected, indicating that this agent did not interact with voltage-activated C a  2 +  channels. DM, at a concentration of 50 uM, reduced high [K*]-  -V-  evoked responses in both neuronal types, showing this agent acted to block neuronal voltage-activated C a  2 +  channels.  Cell viability assays showed enantioselective neuroprotection with (-)- and  (+)-p-  cyclazocine. Hippocampal or cortical neurons were exposed to 1 mM NMDA for 24 hr and the number of surviving neurons following this treatment were compared to the number of neurons before the toxic NMDA exposure. When present during the NMDA insult, (-)-p-cyclazocine protected both hippocampal and cortical neurons, with 50% neuroprotection being achieved near 1 uM. The (+)-enantiomer was weaker as a neuroprotectant, with < 50 % protection at a concentration of 10 uM. The specificities of (-)-p-cyclazocine and DM were further assessed by studying voltage-activated N a and K* currents in DRG neurons and cardiac myocytes. There +  was no detectable effect of either (-)-p-cyclazocine (5 uM) or DM (50 uM) to alter Na* or K* currents in neurons; (-)-p-cyclazocine (5 uM) had no effect on Na* or K* currents in cardiac myocytes. However, DM (50 uM) inhibited both Na* and K* currents in cardiac myocytes, with use-dependent Na* current reduction suggesting DM block of open Na* channels. The results indicate that (-)-p-cyclazocine is a highly potent and selective blocker of NMDA ion channels, with a concomitant neuroprotective capacity. This compound is hence suggested as a possible therapeutic agent for conditions requiring CNS neuroprotection. While DM is an uncompetitive NMDA antagonist, its relative non-selectivity, as demonstrated by actions against neuronal C a * channels and 2  cardiac Na* and K* channels, warrants caution with regard to its use at doses above those required for its antitussive effect and hence may limit its clinical application as a neuroprotectant.  - vi -  TABLE OF CONTENTS  ABSTRACT  TABLE OF CONTENTS  LIST OF FIGURES  LIST OF TABLES  ACKNOWLEDGMENTS  DEDICATION  1. INTRODUCTION  1 . 1 . Excitotoxicity and glutamate 1.1.1. Glutamate release in ischemic brain 1.1.2. Glutamate receptors 1.1.2.1. Kainate and AMPA receptors 1.1.2.2. L-AP4 receptors 1.1.2.3. Metabotropic glutamate receptors 1.1.2.4. NMDA receptors 1.1.3. Regulatory sites of the NMDA receptor-ion channel complex 1.1.3.1. Agonist recognition site 1.1.3.2. Glycine coagonist site 1.1.3.3. Magnesium  - vii 1.1.3.4. Zinc  8  1.1.3.5. Polyamine Site(s)  9  1.1.3.6. Redox sites  10  1.1.3.7. Steroids  11  1.1.3.8. Inhibition by decreasing pH  11  1.1.3.9. Intracellular phosphorylation  12  1.1.3.10. The PCP site  14  1.2. Mechanism of excitotoxicity  15  1.2.1. Initial phase  15  1.2.2. Delayed phase  16  1.2.2.1. Role of calcium 1.2.3. Involvement of NMDA receptors  1.3. Pharmacology of excitotoxicity 1.3.1.1. The concept of "delayed rescue" 1.3.2. Uncompetitive NMDA antagonists  17 18  18 19 20  1.3.2.1. Opiates  21  1.3.2.2. Antitussives  22  1.3.2.3. Sigma ligands  23  1.3.3. (-)-p-cyclazocine  24  1.3.4. Dextromethorphan  25  1.3.5. L-687,384  25  1.4. Experimental approach 1.4.1. Rationale  26 26  1.4.1.1. Blood-brain barrier  26  1.4.1.2. Lack of toxicity  27  1.4.1.3. Selectivity  27  - VIII-  1.4.1.4. Functional efficacy  28  1.4.2. Hypothesis  28  1.4.3. Tissues employed  29  1.4.3.1. Cultured neurons  29  1.4.3.2. CNS areas used  29  1.4.3.3. Cardiac myocytes  31  1.4.4. Strategy  2. M A T E R I A L S A N D M E T H O D S  32  33  2.1. Tissue culture  33  2.2. Elcctrophysiology  34  2.2.1. Patch clamp configurations  35  2.2.1.1. Outside-out patches  36  2.2.2. Single-channel patch clamp procedure  37  2.2.2.1. Pipette preparation  37  2.2.2.2. The recording bath  38  2.2.2.3. Pipette mounting  39  2.2.2.4. Patch formation  39  2.2.2.5. Patch clamp recording, data acquisition, and storage  40  2.2.2.6. Perfusion of solutions  41  2.2.2.7. Data analysis  42  2.2.2.8. Identification of unitary currents through NMDA ion channels  43  2.2.2.9. Statistics  45  2.3. Microspectrofluorimetry  45  2.3.1. Fura-2 experiments  45  2 . 3 . 1 . 1 . Fura-2 loading  46  - ix 2.3.1.2. Measurement of fura-2 fluorescence  47  2.3.1.3. Calculation of [Ca ];  48  2.4. Neuroprotection experiments  49  2+  2.4.1.1. Statistics  51  2.5. Temperature studies  51  2.6. Experiments with voltage-gated Na and K currents +  +  53  2.6.1. Preparation and use of DRG neurons  53  2.6.2. Isolation of rat ventricular myocytes  53  2.6.3. Whole-cell electrophysiology  54  2.6.3.1. Macroscopic recording  54  2.6.3.2. Data analysis  55  3. R E S U L T S  56  3.1. Organization  56  3.2. Electrophysiology  58  3.2.1. Drug actions on amplitudes of unitary NMDA currents and channel conductance in hippocampal neurons  58  3.2.1.1. Amplitudes of unitary NMDA currents and channel conductance in control solution  58  3.2.1.2. Effects of the compounds on unitary NMDA current amplitudes and channel conductance  65  3.2.2. Summary of drug effects on unitary NMDA current amplitudes and channel conductance in hippocampal neurons  74  3.2.3. Drug actions on the kinetics of unitary NMDA currents in hippocampal neurons  75  3.2.3.1. Mean open time of single NMDA ion channels in control solution  75  3.2.3.2. Effects of the compounds on mean open time  76  3.2.3.3. NMDA ion channel opening frequency in control solution  82  - X 3.2.3.4. Effects of the compounds on channel opening frequency  82  3.2.3.5. NMDA ion channel open state probability in control solution  85  3.2.3.6. Effects of the compounds on open state probability  85  3.2.4. Summary of drug effects on unitary NMDA kinetics in hippocampal neurons  87  3.2.5. Consideration of mechanism of uncompetitive antagonist action  87  3.2.5.1. Lack of drug effects on unitary NMDA ion channel conductance  88  3.2.5.2. Decrease of mean open time and mean opening frequency by the compounds  89  3.2.5.3. Applicability of open channel blockade model  89  3.2.6. Analysis of data using an open channel blockade scheme  90  3.2.6.1. Determination of on-rate constants  90  3.2.6.2. Determination of off-rate constants  97  3.2.7. Drug actions on unitary NMDA current amplitude and channel conductance in cortical neurons 3.2.7.1. Amplitudes of unitary NMDA currents and channel conductance in control solution  103 104  3.2.7.2. Effects of (-)-P-cyclazocine and DM on unitary NMDA amplitudes and channel conductance 104 3.2.8. Summary of drug effects on unitary NMDA amplitudes and channel conductance in cortical neurons 115 3.2.9. Drug effects on channel kinetics in cortical neurons  117  3.2.9.1. Effects of (-)-p-cyclazocine and DM on unitary NMDA mean open time  117  3.2.9.2. Effects of (-)-p-cyclazocine and DM on unitary NMDA opening frequency  120  3.2.9.3. Effects of (-)-P-cyclazocine and DM on unitary NMDA channel open probability  123  3.2.10. Summary of drug effects on kinetics of unitary NMDA currents in cortical neurons  126  3.2.11. Applicability of open channel block model in cortical neurons  126  3.2.12. Analysis of cortical neuron data using an open-channel block scheme  127  3.2.12.1. Determination of on-rate constants  127  3.2.12.2. Determination of off-rate constants  127  3.2.13. Conclusions regarding open-channel block analysis in both neuronal types  134  3.2.14. Biophysical probes: (i) studies with Mg * in hippocampal neurons  135  2  3.2.14.1. Interaction of DM with the Mg * site 2  135  - xi 3.2.15. Biophysical probes: (ii) temperature studies with outside-out patches from cortical neurons  137  3.2.15.1. Dependence of unitary NMDA current amplitudes on temperature in control solution  137  3.2.15.2. Dependence of NMDA ion channel kinetics on temperature in control solution  141  3.2.15.3. Effects of DM on temperature modulation of channel behaviour  143  3.2.16. Summary of the use of biophysical probes to elucidate drug actions  3.3. Microspectrofluorimetric studies  143  144  3.3.1. Uncompetitive inhibition of NMDA responses in hippocampal neurons 2+ 3.3.1.1. p-cyclazocine blockade of NMDA-evoked [Ca ]i responses: use-dependence 2+ 3.3.1.2. DM effects on NMDA-evoked [Ca ]i responses 2+  3.3.1.3. L-687,384 effects on NMDA-evoked [Ca ]i responses  145 150 150  3.3.2. Drug effects on calcium responses to raised K in hippocampal neurons +  2+ 3.3.2.1. Effects of (-)-p-cyclazocine on [Ca ]i reponses to raised K 2+ 3.3.2.2. Effect of DM on [Ca ]i responses to raised K  145  +  153 153  153  +  3.3.3. Drug effects on non-NMDA responses in hippocampal neurons  156  3.3.3.1. Effects of (-)-p-cyclazocine on [Ca ]; responses to non-NMDA agonists  156  3.3.3.2. Effects of DM on [Ca ] responses to non-NMDA agonists  157  3.3.4. Drug blockade of NMDA ion channels in cortical neurons 2+ 3.3.4.1. P-cyclazocine blockade of NMDA-evoked [Ca ]{responses 2+ 3.3.4.2. D M blockade of NMDA-evoked [Ca ]i responses 3.3.5. Drug effects on calcium responses to raised K in cortical neurons  157  2+  2+  ;  +  2+ 3.3.5.1. Effect of (-)-P-cyclazocine on [Ca ]i reponses to raised K 2+ 3.3.5.2. Effect of D M on [Ca ]i responses to raised K  +  +  3.3.6. Drug effects on non-NMDA responses in cortical neurons  157 161 164 164 164 164  3.3.6.1. Effects of (-)-P-cyclazocine on [Ca \, responses to non-NMDA agonists  164  3.3.6.2. Effects of DM on [Ca ]; responses to non-NMDA agonists  169  2+  2+  3.4. Neuroprotection studies  169  - XII -  3.4.1. (-)-P-cyclazocine as a candidate neuroprotectant  169  3.4.2. Neuroprotection studies with hippocampal neurons  170  3.4.2.1. Excitotoxicity produced by NMDA in vitro  170  3.4.2.2. Neuroprotection by (-)-p-cyclazocine in hippocampal neurons  173  3.4.2.3. Effect of (+)-P-cyclazocine in hippocampal neurons  173 176  3.4.3. Neuroprotection studies with cortical neurons 3.4.3.1. Effects of NMDA only  176  3.4.3.2. Neuroprotection by (-)-P-cyclazocine in cortical neurons  176  3.4.3.3. Effects of (+)-p-cyclazocine in cortical neurons  177  3.S. Actions of the compounds on voltage-gated ion channels 3.5.1. Macroscopic currents in dorsal root ganglion (DRG) cell line neurons 3.5.1.1. Properties and pharmacology of I  177 177 180  K  3.5.1.2. Effects of D M and (-)-P-cyclazocine on I  K  180  3.5.1.3. Properties and pharmacology o f l N a in neurons  183  3.5.1.4. Effects of D M and (-)-p-cyclazocine on I  188  NA  3.5.1.5. Conclusions regarding drug effects on macroscopic currents in neurons 3.5.2. Macroscopic currents in ventricular myocytes  188 189  3.5.2.1. Properties and pharmacology of I in ventricular myocytes  189  3.5.2.2. Effects of (-)-p-cyclazocine on 1^  192  3.5.2.3. Effects of D M on lu,  193  TO  3.5.2.4. Properties and pharmacology of I  NA  in ventricular myocytes  196  3.5.2.5. Effect of (-)-p-cyclazocine on IN in ventricular myocytes  196  3.5.2.6. Effect of D M on I in ventricular myocytes  196  3.5.2.7. Conclusions regarding drug effects on macroscopic currents in ventricular myocytes  197  8  NA  4. DISCUSSION  199  - XIII-  4.1. Neuroprotection by (-)-fi-cyclazocine  199  4.1.1. NMDA ion channel blockade by (-)-j)-cyclazocine results in neuroprotection  199  4.1.2. Actions of (-)-p-cyclazocine relevant to neuroprotection  200  4.1.2.1. Open channel block and the on-rate constant  201  4.1.2.2. The off-rate constant  203  4.1.2.3. Potency of (-)-P-cyclazocine and comparison with other agents  204  4.1.2.4. Inhibition of NMDA-evoked [Ca ] responses  204  4.1.2.5. Trapping of antagonist within closed channels  205  4.1.2.6. Potent neuroprotection with p-cyclazocine is stereospecific  206  4.1.2.7. Non-opiate mechanism  208  4.1.2.8. Predictive power of model  209  4.1.2.9. Selectivity for NMDA channels vs. other channels  210  2+  ;  4.1.3. (-)-P-cyclazocine is a candidate neuroprotectant  4.2. NMDA antagonism by DM  212  213  4.2.1. Interactions of neuroprotectant concentrations of DM with NMDA ion channels  213  4.2.2. Actions of DM relevant to neuroprotection  214  4.2.2.1. Alteration in NMDA ion channel kinetics . 4.2.2.2. Open channel block by DM and the forward rate constant  214 214  4.2.2.3. Voltage-dependence of on-rate constant  215  4.2.2.4. High off-rate constant for DM and the implication for clinical use  216  4.2.2.5. The nature of the DM binding site: biophysical studies  216  4.2.2.6. DM reduction of NMDA-evoked [Ca ]i responses  217  4.2.2.7. DM actions on voltage-activated Ca channels  218  4.2.2.8. Lack of effect on non-NMDA ion channels  219  4.2.2.9. Lack of DM actions on voltage-gated ion channels in neurons and cardiac myocytes  219  2+  2+  4.3. NMDA antagonism by L-687,384  221  - xiv4.3.1. Interaction of L-687,384 with NMDA ion channels  221  4.3.1.1. Open-channel block of NMDA ion channels  221  4.3.1.2. Fast kinetics of block  221  4.3.1.3. Relationship between open channel block andCTeffects  222  5. S U M M A R Y  6. R E F E R E N C E S  224  230  -XV -  LIST OF FIGURES Figure  Title  Page  1  Unitary NMDA currents in hippocampal neurons  60  2  Current-voltage relation for the patch shown in Figure 1 in control solution  63  3  Unitary currents before and after exposure to (-)- or (+)-P-cyclazocine in hippocampal neurons  67  4  Amplitude distributions in the absence or presence of (-)- or (+)-p-cyclazocine in hippocampal  69  neurons 5  Current-voltage relation for the patch shown in Figures 3 and 4 exposed to 1 uM (-)-P-cyclazocine  71  6  Open time distributions in the absence and presence of (-)-or (+)-p-cyclazocine in hippocampal  78  neurons 7  Opening frequencies of NMDA ion channels in the presence of (-)- or (+)-p-cyclazocine in  81  hippocampal neurons 8  Open state probabilities of NMDA ion channels in the presence of (-)- or (+)-p-cyclazocine in  84  hippocampal neurons 9  Determination of onward rate constants for (-)- or (+)-p-cyclazocine in hippocampal neurons  92  10  Determination of onward rate constants for DM in hippocampal neurons  94  11  Determination of onward rate constants for L-687,384 in hippocampal neurons  96  12  Determination of off-rate constant k_ in hippocampal neurons  99  13  Unitary NMD A currents in cortical neurons  106  14  Current-voltage relation for the patch shown in Figure 13 in control solution  108  15  Unitary currents after exposure to (-)-p-cyclazocine in cortical neurons  110  16  Amplitude distributions in the absence and presence of (-)-p-cyclazocine in cortical neurons  112  17  Current-voltage relation for the patch shown in Figures 13 -16 during exposure to 200 nM (-)-P-  114  2  cyclazocine 18  Open time distributions in the absence and presence of (-)-P-cyclazocine in cortical neurons  119  19  Opening frequencies of NMDA ion channels in the presence of (-)-p-cyclazocine in cortical neurons  122  - xvi -  Figure 20  Title Open state probability of NMDA ion channels in the presence of (-)-p-cyclazocine in cortical  Page 125  neurons 21  Determination of the onward rate constant for (-)-p-cyclazocine in cortical neurons  129  22  Determination of the onward rate constant for DM in cortical neurons  131  23  Determination of off-rate constant k_2 in cortical neurons  133  24  Dependence of unitary NMDA currents on temperature  139  25  Attenuation of NMDA-evoked rises in [Ca ]; by P-cyclazocine in hippocampal neurons  147  26  Selectivity of (-)-p-cyclazocine for NMDA responses in hippocampal neurons  152  27  Selectivity of DM for NMD A responses in hippocampal neurons  155  28  Reduction of 20 uM NMDA-evoked [Ca ]i responses by P-cyclazocine in cortical neurons  159  29  Reduction of 20 uM NMDA-evoked [Ca ]i responses DM in cortical neurons  163  30  Selectivity of 5 uM (-)-p-cyclazocine in cortical neurons  166  31  Responses to raised K and non-NMDA agonists in the absence and presence of DM in cortical  24  2+  2+  +  168  neurons 32  Protection against NMDA excitotoxicity by p-cyclazocine in hippocampal neurons  172  33  Stereospecific neuroprotection by p-cyclazocine in cortical neurons  175  34  TEA-sensitive delayed rectifier in DR'G neurons  179  35  Neuronal IK in the absence and presence of DM  182  36  TTX-sensitive sodium current in DRG neurons  185  37  Neuronal I in the absence and presence of DM  187  38  Actions of D M on cardiac I*,  191  39  Actions of D M on cardiac IN.  195  NA  -XVII -  LIST OF T A B L E S Table 1  Title Unitary  N  M  D  current amplitudes  A  (INMDA),  conductances  Page ( G ) ,  and reversal potentials  (  V  W  )  for  73  control and in the presence of the compounds indicated, as measured in outside-out patches from hippocampal neurons 2  Unitary  N  M  D  current amplitudes (INMDA), conductances  A  ( G ) ,  and reversal potentials (V ) for m  116  control and in the presence of the compounds indicated, as measured in outside-out patches from cortical neurons 3  Qio values for channel amplitude in the absence and presence of DM  140  4  Qio values for channel mean open times in the absence and presence of DM  142  5  Reduction of responses to  by P-cyclazocine in hippocampal neurons  149  6  Reduction of  N  M  D  A  N  M  D  A  responses by P-cyclazocine in cortical neurons  160  -XVIII-  ACKNOWLEDGMENTS I would like to express my gratitude to my Research Supervisor, Dr. James G. McLarnon for his encouragement and guidance. I am indebted to Drs. David Godin, Casey van Breemen, and Alex Goumeniouk for their participation in my thesis committee, and for their careful reading and much-needed comments for this thesis. I am grateful to the faculty and staff members of the Department of Pharmacology & Therapeutics for the use of their facilities and their assistance. I am indebted to Ms. Monika Grunert, whose contribution toward the cell cultures was invaluable.  I would like to thank Mr. Zhongxiang Huang for his technical  participation in the excitotoxicity experiments. I am very grateful to Dr. John Church for the  use  of  his  compounds,  his  expertise,  and  his  facilities  for  the  microspectrofluorimetric experiments. Finally, the financial support of the Canadian Heart and Stroke Foundation is greatly appreciated.  - xix DEDICATION This thesis is dedicated to my mother, Lynn, my father, Ronald, and my brother, Michael, without whose love, encouragement, and support none of my studies would have been possible.  - 1 -  1.  INTRODUCTION 1.1.  Excitotoxicity and glutamate  The involvement of glutamate receptors in the pathogenesis of neuronal injury following cerebral ischaamia/hypoxia has been acknowledged for a number of years. Observations of neuronal necrosis following systemic administration of acidic amino acids, including L-glutamate (or its salt monosodium glutamate, or MSG), L-aspartate, and L-cysteine, were the first controlled experiments to demonstrate the debilitating effects of acidic amino acids on the CNS (Olney, 1969; Olney and Sharpe, 1969; Olney et al., 1971).  Examination of electron microscope data showed the most sensitive  areas to be those most exposed to the systemic route of administration, specifically, the arcuate layer of the hypothalamus and the inner layer of the retina. This suggested that the damage was a result of a direct effect of the administered amino acids on the tissue.  1.1.1. Glutamate release in ischemic brain Evidence has accumulated to implicate the involvement of L-glutamate in neuronal brain damage in vivo, particularly where there is a brief interruption of oxygen supply to the damaged area, such as in stroke, head trauma, or hypoxia (Rothman, 1984).  Under these conditions,  extracellular  L-glutamate concentrations  rise  dramatically to supraphysiologic levels (Benveniste et al., 1984). When oxygen supply fails to meet demand, cellular energy metabolism is compromised, and processes which depend on cellular ATP stores are thus impaired.  For instance, the loss of  - 2 energy required to maintain cell resting potential results in neuronal depolarization, which in turn releases L-glutamate into the synapse; energy-requiring  glutamate  transport from the synapse is also affected. This constitutes part of a "feed-forward" cycle, in which glutamate released into the synapse (and not taken up) would therefore produce depolarization and further glutamate release from the post-synaptic cell (see Choi, 1988a,b). The net result is a dramatic increase in the extracellular concentration of L-glutamate (Benveniste et al., 1984; Rothman and Olney, 1986). Neurons that lyse upon death and release their cellular contents into the neuropil also contribute to. the increased glutamate concentration in such conditions; the concentration of glutamate in neuronal tissue is in the millimolar range (Curtis and Johnston, 1974; Choi, 1988a) and is thus a large pool of glutamate that would result in a significant rise in glutamate concentration in the neuropil upon cell lysis. As well, there is evidence that astrocytes, which also contain high levels of glutamate, are also possible sources of L-glutamate, either as physiologically released L-glutamate or that arising from lysing cells (Dawson et al., 1995; Kimelberg et al., 1995; Jeftinija et al., 1996).  1 1 2 . Glutamate receptors Glutamate has long been recognized as the principal excitatory neurotransmitter in the mammalian brain. Re-evaluation of a series of experiments performed in the early 1960's (Curtis and Watkins, 1962) showed that not only was L-glutamate (or its analog L-aspartate) acting on specific receptors, but that (although not realized at the time) there were several subtypes of glutamate receptor, each signalling the neuron in different ways. There are 5 subtypes of glutamate receptor currently accepted:  3  ionotropic receptors (NMDA, kainate, and A M P A (a-amino-2,3-dihydro-5-methyl-3-oxo-  - 3 4-isoxazole-propanoic acid) receptors), a presynaptic receptor, and a metabotropic receptor. The following section is a brief description of the properties of the known glutamate receptor subtypes.  For more extensive reviews concerning glutamate  receptors, see Mayer and Westbrook (1987b),  Monaghan et  al. (1989),  and  Collingridge and Lester (1989). 1.1.2.1. Kainate and AMP A receptors Two subtypes of the glutamate receptor are coupled to ion channels that are non-selectively permeable to Na* and K* and their activation results in neuronal depolarization (Ascher and Nowak, 1988a). These are the kainate receptors and the A M P A receptors, both named for the compounds which preferentially activate that subtype. The latter (AMPA) subtype was originally known as the quisqualate receptor but was renamed due to AMPA's higher selectivity for that subtype.  The chief  differences between A M P A and kainate receptors are their distribution in the CNS, the selectivity of the two subtypes for different agonists, and the membrane conductance changes mediated by either subtype (Ascher and Nowak, 1988a). Both of the above receptor subtypes are mediators of fast synaptic transmission in the mammalian C N S (Lambert ef a/., 1989).  1.1.2.2. L-AP4 receptors The  L-AP4  (L-2-amino-4-phosphonobutanoate)  receptor  subtype  is  an  autoreceptor that controls glutamate release through a negative feedback mechanism. This receptor subtype is believed to be located presynaptically on glutamatergic terminal boutons (Monaghan et ai, 1989). Because of its presynaptic location, little is  - 4 known about the mechanism through which the L-AP4 receptor limits glutamatergic synaptic activity.  Additionally, progress into characterizing the L-AP4 receptor has  been hampered by the fact that L-AP4 itself can activate certain metabotropic glutamate receptors (Mattson and Mark, 1996; see below). J.].2.3.Metabotropic  glutamate receptors  The metabotropic glutamate receptor is unique among glutamate receptor subtypes in that its activation results not in ion channel opening but rather the triggering of second messenger systems that alter the internal biochemical environment of the cell.  These include generation of intracellular second messengers, such as  inositol 1,4,5-triphosphate and diacylglycerol, that in turn intracellularly regulate the excitability of the neuron (Sugiyama et al., 1987; Challiss et al., 1994).  Molecular  sequencing work has revealed the existence of at least seven metabotropic receptor subtypes, termed mgluRI to 7, and this family of metabotropic glutamate receptors has now emerged as important regulators of cell behaviour (Bruno et al., 1996; Mattson and Mark,  1996).  quisqualate,  Metabotropic glutamate and  synthetic  amino  receptors are activated by glutamate, acid  analogs  such  as  1,3-  aminocyclopentanedicarboxylic acid (frans-ACPD) (Bruno et al., 1996; Monaghan et al., 1989).  1.1.2.4.NMDA receptors  The remaining subtype, the NMDA receptor, is perhaps the most extensively studied subtype of the glutamate receptor family. A major characterizing feature of the NMDA receptor is that it is coupled to an ion channel that is highly permeable to  - 5 calcium (Dingledine, 1983; MacDermott etal., 1986) while exhibiting voltage-dependent block by Mg * (Nowak et al., 1984; Jahr and Stevens, 1990). The receptor itself is 2  thought to be a heteroligomeric complex consisting of a subunit termed NR1 in association with one of another set of subunits, termed NR2A - E (for reviews, see Mattson and Mark, 1996; McBain and Mayer, 1994). The unique properties of NMDA receptors mentioned above contribute to the unique physiology of the NMDA receptor and allow it to participate centrally in a variety of functions, including long-term potentiation (the process believed to be the cellular correlate of long-term memory; Bliss and Collingridge, 1993), synaptic development and plasticity (Constantine-Paton et al., 1990), and the generation of rhythmic motor activity (Headley and Grillner, 1990). NMDA receptors have been implicated in a number of glutamate-related pathologies, such as epilepsy (Dingledine et al. 1990), neurodegenerative disorders (Choi, 1988b; Meldrum and Garthwaite, 1990; Danysz et al., 1995), and excitotoxicity (see below), and it appears that all these conditions have as a common factor the high intracellular calcium levels induced by excessive NMDA receptor activation.  1.1.3. Regulatory sites of the NMDA receptor-ion channel complex The NMDA receptor-ion channel complex is a complicated entity, with several sites at which ligands can act to modify the behaviour of the channel. It has regulatory sites than any known receptor, with new sites still being discovered. Investigators hope to take advantage of this multi-faceted  regulation, in that many avenues to  pharmacological manipulation of the activity of the NMDA system are thus potentially available.  -  6  -  1.1.3.1. Agonist recognition site  Compounds that occupy the receptor site where L-glutamate or NMDA attach and which do not possess intrinsic excitatory activity are by definition competitive antagonists. The majority of compounds in this class have sufficient charge on the molecule to prevent entry into the central nervous system (CNS) through the bloodbrain barrier.  Clinical trials of some agents that are sufficiently lipophilic to pass  through the blood-brain barrier are currently under way (Lipton, 1993; Koroshetz and Moskowitz, 1996). However, C N S toxicity is a risk, since simple competitive inhibition of NMDA activity may impair synaptic transmission to an unacceptable degree (Grotta, 1994). Furthermore, it should be noted that the effects of competitive inhibition can be overcome by increasing concentrations of agonist.  J. J. 3.2. Glycine coagonist site  The requirement of ambient levels of glycine, at the mid-nanomolar level, in the extracellular medium for expression of NMDA activity has led to the discovery of a coagonist site for glycine on the NMDA receptor-ion channel complex (Johnson and Ascher, 1987; Kleckner and Dingledine, 1988; Reynolds and Miller, 1988). The identity of this site was initially cloaked by the ubiquitous presence of glycine, since this amino acid can be present at micromolar levels even in "purified" distilled water. The subsequent development of antagonists and partial agonists for this site, such as HA966 (R-(+)-3-amino-1-hydroxypyrrolid-2-one;  Kemp et al., 1988; Henderson et al.,  1990), showed that NMDA activity could be blocked by antagonizing the effects of glycine at this site. It is believed that the recognition site for glycine on the NMDA  - 7receptor is located near the recognition site for glutamate on the extracellular loop of the NR1 subunit (Hirai et al., 1996), and that glycine acts by regulating the level of desensitisation of the NMDA receptor-ion channel complex (Mayer et al., 1989). The receptor for glycine on the NMDA receptor-ion channel complex is different from that associated with the synaptic glycine receptor coupled to a chloride channel. First, the glycine site on the NMDA receptor has a far greater affinity for glycine than does the inhibitory receptor, with the Ko of the former being near 100 nM (134 nM; Manallack et al., 1990) and the Ko of the latter being close to 20 pM (24 pM; Graham et al., 1985). Second, the pharmacology of the two sites is not the same.  The most notable  pharmacological difference between the two types of glycine receptor is the lack of sensitivity of the NMDA-associated site to strychnine (Johnson and Ascher, 1987), which potently antagonizes the inhibitory glycine receptor (Curtis et al. 1968; Graham et al. 1985); for this reason, the NMDA-associated glycine coagonist site is commonly referred to as the strychnine-insensitive glycine site.  Modulation of NMDA activity  through this site can provide neuroprotection (Newell et al., 1995), and currently a number of strychnine-insensitive glycine receptor antagonists are being tried in the clinical setting for use in stroke therapy (Danysz et al., 1995).  1.1.3.3.Magnesium  The blockade of the NMDA channel by Mg * confers voltage-dependent 2  properties on the NMDA-activated membrane conductance (Nowak et al., 1984; Mayer et al., 1984; Mayer and Westbrook, 1987a). The normal concentration of Mg * in the 2  extracellular fluid is sufficient to prevent ion flux through NMDA channels at resting  - 8 potential, whereas at depolarized potentials the block by M g  2+  is relieved and current  flow through NMDA ion channels proceeds (Ault et al., 1980). In this sense, NMDA activity serves to enhance the effects of other forms of excitation, such as activation of non-NMDA glutamate channels. Alternatively, NMDA activity is engineered to "kick in" only when there is sufficient excitatory input from other sources to relieve the Mg * 2  block. The site at which M g  2+  exerts its blocking action is deep within the ion channel;  voltage-dependence occurs because the charged Mg * ion must traverse a significant 2  portion of the membrane electric field in order to reach its site (Ascher and Nowak, 1988b). Attempts to exploit the inhibitory effects.of Mg * on NMDA activity for clinical 2  use in conditions such as epilepsy should be appraoched with caution, since in most pathological states in the CNS, neurons are depolarized to potentials where Mg * has 2  little effect. As well, the non-specificity of Mg *, particularly with respect to its blockade 2  of C a * channels (Hess and Tsien, 1984; Hess et al., 1986; Lansman et al., 1986), 2  complicates its effects.  However, preliminary clinical trials dealing with the clinical  application of Mg * in epilipsy have shown promise in the lack of adverse effects, and 2  phase 3 trials are currently being planned (Muir and Lees, 1995). To date, there are no known agents besides Mg * that act at the magnesium site and which can be exploited 2  for use in either the laboratory or clinical settings.  U.3.4.Zinc Zn * ions non-competitively inhibit NMDA activity, possibly by preventing the 2  ability of agonists to gate open the channel (Westbrook and Mayer, 1987; Reynolds and Miller, 1988; Forsythe et ai, 1988).  The lack of voltage-dependence of Zn * 2  - 9 actions suggests that its binding site is located on the extracellular surface of the channel protein.  Ionic zinc is present in the nerve terminals of central excitatory  neurons (Frederickson et al., 1982; Danscher et al., 1985) and is co-released with glutamate from the presynaptic neuron into the synaptic cleft (Assaf and Chung, 1984; Howell et al., 1984; Charton et al., 1985). It is believed that Z n of endogenous glutamate-related NMDA activity. histamine,  has  recently  been  shown  to  2+  serves as a regulator  Another endogenous compound,  potentiate  responses  to  NMDA  at  concentrations < 0.5 mM; since this action is voltage-independent, the possibility exists that histamine acts through the Z n Zn  2+  2+  site, or close to it (McBain and Mayer, 1994). The  site on the NMDA receptor-ion channel complex has yet to be exploited as an in  vivo mitigator of NMDA activity; however, further research, particularly with repect to histamine's effects at this site (see above), may reveal any clinical significance associated with manipulation of Z n  2+  actions on NMDA activity.  1.1.3.5.Polyamine Site(s)  Another important area concerns the regulation of NMDA activity by polyamines such as spermine and spermidine.  Binding experiments have shown that, in the  presence of glutamate, spermine and spermidine enhance the binding of radiolabelled uncompetitive NMDA antagonists such as [ H]TCP and [ H]MK-801, but not [ H]glycine 3  3  3  or the competitive antagonist [ H]CPP, suggesting these compounds promote channel 3  opening via sites distinct from the recognition sites for either glutamate or glycine (Ransom and Stec, 1988; Carter et al., 1990).  In whole-cell electrophysiological  recordings, these compounds showed a complex mode of action, with an enhancement  - 10of NMDA responses being observed at low (< 1 mM for spermine) concentrations and antagonism being the effect of higher concentrations (Benveniste and Mayer, 1993). The two effects may be mediated by separate sites on the NMDA ion channel, as other polyamines are capable only of the block at higher concentrations without producing potentiation at the lower concentrations (McBain and Mayer, 1994).  The voltage-  dependence of the NMDA-modulating properties of polyamines suggests the sites of attachment are located within the channel pore.  It has been suggested that some  aspects of histamine modulation of NMDA receptor activity are mediated via a polyamine binding site, including the antagonism produced by high (> 0.5 mM) concentrations of histamine (Bekkers, 1993; Vorobjev etal., 1993). 7.7.3.6.Redox sites  Redox modulation of the NMDA receptor-ion channel complex is a recently discovered phenomenon that promises to reveal much about how NMDA activity is endogenously regulated from the outside of the neuron. The NMDA complex is similar to many other transmitter-gated ion channels in this respect.  Reduction of NMDA  receptors with agents such as dithiothreitol (DTT) enhances their activity whereas oxidation, as with 5,5'-dithiobis-2-nitrobenzoic acid (DTNB), inhibits their (Gozlan and Ben-Ari, 1995).  activity  Both the former and latter agents are nonpermeant  through cell membranes and are therefore affecting redox sites located on the extracellular side of the receptor. The fact that either reducing or oxidizing the receptor changes its activity suggests the resting native NMDA receptor is in an equilibrium state between a fully reduced form and a fully oxidized form (Aizenman et al., 1989).  -11Possible endogenous compounds that may be active in affecting NMDA receptors in this way are thiol derivatives such as cysteine, homocysteine, glutathione, lipoic acid, and dihydrolipoic acid, ascorbate, and free radicals such as nitric oxide (NO) (Lipton et al., 1993; Tang and Aizenman, 1993; Gozlan and Ben-Ari, 1995). 1.1.3.7.Steroids  Steroids such as pregnenolone have been shown to directly enhance NMDA activity (Wu et al., 1991; Irwin et al., 1992) in a manner not related to steroid effects on gene expression (Bowlby, 1993). The site through which this effect is mediated is most likely accessed via the lipid bilayer, as suggested by both the kinetics of steroid action and the observation that external application of pregnenolone can affect NMDA channel behaviour as recorded in both cell-attached and outside-out patches (Bowlby, 1993).  1.1.3.8. Inhibition by decreasing pH  Increasing the acidity of the extracellular solution inhibits NMDA responses (Traynelis and Cull-Candy, 1990) while increasing the alkalinity increases NMDA activity (Giffard et al., 1992). This action is voltage-independent and non-competitive. The modulation of NMDA activity by acidity may represent a compensatory negative feedback mechanism that would come into play during the fall in pH observed after periods of excitatory synaptic activity.  Furthermore, since the apparent PKA of the  NMDA receptor-ion channel complex is quite close to physiological pH {i.e., near pH 7.0; Traynelis and Cull-Candy, 1990, 1991), NMDA responses are necessarily quite sensitive to small changes in extracellular pH which would generally be occurring along  - 12the steep portion of the acid-base curve.  In contrast, AMPA and kainate receptors  show ICso's for acid-mediated inhibition to be near pH 6.0, outside the range of normal physiological pH (Traynelis and Cull-Candy, 1991).  1.1.3.9.Intracellular phosphorylation  Intracellular factors, such as kinases and phosphatases, play an important role in the overall activity of NMDA receptor-ion channels. The phosphorylation states of certain amino acids on the channel protein (or on cytoskeletal elements associated with and possibly playing a regulatory role on NMDA receptors; see Rosenmund and Westbrook, 1993) have a profound effect on the behaviour of NMDA ion channels (Kaczmarek, 1987; Huganir and Greengard, 1990), as well as on the distribution of NMDA receptor subunits between the membrane and intracellular locations (Tingley et al., 1997). In this way, the state of the NMDA receptor may be controlled distally. For instance, generation of secondary messengers by metabotropic glutamate receptors will in turn affect the excitability of the NMDA ion channel by activating protein kinases that phosphorylate certain sites on the cytoplasmic face of the channel (Ben-Ari et al., 1992; Krieger et al., 1996). These kinases include protein kinase C (PKC; Tingley et al., 1997), cAMP-dependent protein kinase (protein kinase A, or PKA; Tingley et al., 1997), tyrosine kinases (Wang and Salter, 1994), and serine/threonine kinases (Tingley et al., 1993).  Serine/threonine phosphatases, such as Ca /calmodulin2+  dependent phosphatase 2B (calcineurin; Lieberman and Mody, 1994) or protein phosphatases 1 and 2A (Wang etal., 1994), have also been shown to play a role in the reguation of NMDA receptor-ion channel complexes, particularly through sites on the  - 13NR2A and NR2B subunits (Menegoz et al., 1995), which also contain the sites for regulation by tyrosine kinases (e.g., PTK pp60 " ; Yu et al., 1997). Sites for P K C and c  s,c  PKA phosphorylation have been found on the NR1 subunit (Tingley et al., 1993; Tingley et al., 1997), and these sites have been suggested to regulate the sensitivity of NMDA receptors to M g  2+  (Chen and Huang, 1992), as well as NR1 subcellular  distribution (see above). PKA has been shown to increase the opening frequencies and mean open times of NMDA-activated ion channels (Greengard et al., 1991). The metabolic state of the neuron can also influence NMDA activity, as kinase and phophatase activities depend on the availability of ATP as a source of organic phosphorous.  In this regard, prolonged whole-cell recording of NMDA receptor-  mediated currents requires the inclusion of an ATP-generating system in the recording pipette, in order to prevent washout or rundown of these currents .that would otherwise occur (MacDonald et al., 1989; Wang et al. 1993). The intracellular enzyme protein kinase C (PKC) is thought to be heavily involved in NMDA receptor-ion channel regulation, for a variety of reasons: rises in intracellular free calcium ([Ca ]j), such as 2+  that produced by NMDA receptor activation, stimulate P K C activity (Gerber  et al.,  1989; Ben-Ari et al., 1992; Krieger et al., 1996), a self-enhancing property that may be relevant to plastic processes such as LTP; activation of metabotropic  glutamate  receptors has been shown to enhance NMDA receptor-mediated responses through a PKC-dependent mechanism (Kelso etal., 1992); and direct activation of P K C activity by phorbol esters potentiates NMDA responses (Gerber et al., 1989).  - 141.1.3.10.The PCP site  An important class of NMDA antagonist, the uncompetitive inhibitors, act through a binding site not associated with the recognition sites for glutamate or glycine. Certain features of the antagonism by the uncompetitive inhibitors and that produced by Mg * 2  suggest the existence of separate and distinct binding sites, with the latter showing properties more consistent with a location deep within the channel pore (MacDonald and Nowak, 1990; MacDonald et ai, 1991). The classical ligand for the uncompetitive antagonist binding site is the dissociative anaesthetic phencyclidine, or P C P (Anis et ai, 1983; Honey et ai, 1985), and the binding locus is hence often referred to as the P C P site.  Ketamine is another well-known example in this class of open-channel  NMDA blockers (Martin and Lodge, 1985; Honey et ai, 1985).  Access to this site  requires the ion channel to be in the open conformational state; consequently, the presence of agonist is a requirement for the action of these agents. Unlike competitive antagonists, the uncompetitive NMDA antagonists show an increasing level of blockade with increasing agonist activity (Parsons et ai., 1992).  The property of use-  dependence, in which the level of blockade increases with subsequent agonist applications, is a characteristic of agents in this class (MacDonald et ai, 1987; MacDonald and Nowak, 1990). Uncompetitive NMDA antagonists hold great promise for therapeutic application, since these agents are selective for highly active ion channels and exert little effect on low, basal levels of activity. The processes in which the NMDA receptor-ion channel complex participate (e.g. synaptic transmission, LTP) would be left relatively intact, while pathophysiological actions mediated by increased levels of agonist, and hence increased NMDA activity, would be selectively targetted.  -15Unlike M g , these agents interact with the blocking site relatively slowly; dissociation 2+  from the channel site occurs on a longer time scale and the channel may even close with the antagonist molecule(s) still trapped within the ion channel (Javitt and Zukin, 1989). It is indeed possible for a given agent to dissociate so slowly from the site that it may, for all intents and purposes, resemble an irreversible blocker. This situation is most clearly demonstrated by MK-801 (Kemp et al.. 1986; Wong and Woodruff, 1986; Foster and Wong, 1987), an anticonvulsant that is not used in humans because of its lengthy duration of action (Lipton, 1993; Rogawski, 1993).  1.2.  Mechanism of excitotoxicity  The early experiments by Olney and colleagues demonstrated a correlation between the excitatory potency of a series of glutamate and aspartate analogues and their potency in producing neuronal injury, although the latter effect required much higher concentrations than what would be expected for simple excitation of central neurons (Olney et al., 1971). This observation led to the conclusion that glutamate receptors are important mediators of the excitotoxic process.  1.2.1. Initial phase The neuronal damage that results from exposure to high concentrations of an excitatory agent, such as L-glutamate or L-aspartate, can be separated into two phases. The first, deemed the initial phase, occurs immediately after exposure to an excitotoxic insult, and has been shown to depend on activation of non-NMDA ion channel-coupled receptors, i.e., kainate or A M P A receptors (Choi, 1987a, 1988a,b; Meldrum and Garthwaite, 1990). The sustained depolarization resulting from overactivation of these receptors produces an excessive influx of Na , Cl", and H 0 (Rothman, 1985; Choi, +  2  - 161987a; Takahashi ef al., 1995). Associated with this is an expansion of intracellular volume, seen under the light microscope as swelling of neuronal somata. The ionic influx thus depends on the compromised resting potential of the neuron; this is corroborated by the ability of high (depolarizing) concentrations of K* to mimick the acute phase of excitotoxicity (Choi, 1987a). If the excitatory stimulus is withdrawn, cells can recover from this phase. However, this recovery depends on the severity of the insult (as cells may burst if the inner volume expands too greatly) and to what degree the second phase (see below) occurs. 1.2.2. Delayed phase The second, or late, phase is separated from the initial phase by a length of time exceeding several hours. It is characterized by a marked degeneration of the neuronal plasma membrane, visible under the light microscope as a "beading" of the cell surface. This phase is dependent on the presence of extracellular calcium (Choi, 1987a; Takahashi, 1995), and the calcium ionophore A23187 can produce a similar pattern of neurodegeneration by allowing a supraphysiological influx of C a  2 +  into the cell (Choi,  1987a). Indeed, it has been shown that the greatest accumulation of C a  2 +  into neurons  in vivo following ischaemic insult occurs in the C N S areas that are most vulnerable to excitotoxic damage (Baumgarten and Zimmerman, 1992).  While the initial and the  delayed phases can both lead to neuronal death, evidence indicates the latter phase is more important at lower levels of toxic excitatory amino acid exposure and may predominate under many pathological conditions (Choi, 1988a).  - 171.2.2.1.Role of calcium Entry of supraphysiologic levels of C a  2 +  serves as a trigger for processes that  eventually lead to disintegration of structural components and loss of control of ionic disposition via leak through the compromised plasma membrane.  C a * serves as an 2  activator of many intracellular enzyme systems (reviewed in Choi 1988a,b; Danysz et al., 1995), for example, (a) the degradative protein calpain 1 (Siman and Noszek, 1988), (b) phospholipases that liberate arachidonic acid, which is in turn acted on by intracellular oxidases to generate superoxide radicals (Chan and Fishman, 1980), and (c) a Ca -activated protease that converts xanthine dehydrogenase to xanthine 2+  oxidase, which in turn produces toxic oxygen-containing free radicals (Dykens et al., 1987). Mitochondrial oxidative phosphorylation can also be impaired by excessively high intracellular C a  2 +  levels, leading to decreased efficiency of energy-dependent C a * 2  transporters (Siesjo, 1988; Choi, 1988b).  Additionally, elevated intracellular calcium  can itself stimulate further increases in its own concentration, by (i) reversing the Na /Ca +  2+  exchanger, allowing C a * to enter the cell via this pump (Nachsen et al., 2  1986), (ii) generation of inositol 1,4,5-triphosphate, which can liberate C a * from 2  intracellular stores (Berridge, 1993), (iii) activation of endoplasmic reticulum (ER)based ryanodine-sensitive receptors causing C a * release from the E R (Mody and 2  MacDonald, 1995), and (iv) acting with diacylglycerol to activate PKC, which enhances the activity of voltage-gated calcium channels, NMDA channels, and other channels (Kaczmarek, 1987; Connor etal., 1988).  - 18-  1.2.3. Involvement of NMDA receptors The observation that the late phase of glutamate-induced neurotoxicity is antagonized by A P V (Choi, 1988a) has revealed the central role that NMDA receptors play in excitotoxicity.  Exposure of neurons in vitro to high levels of glutamate in the  presence of A P V abolishes the late excitotoxic phase while leaving the initial phase intact (Choi et al., 1988). During periods of excessive glutamate activation, C a the neuron through NMDA ion channels, especially when M g  2+  2 +  enters  block is relieved by  depolarization via activation of non-NMDA receptors. Concurrently, intracellular free calcium ([Ca ]j) rises dramatically, some from direct entry via NMDA ion channels, and 2+  some from other sources, such as Ca -induced C a 2+  organelles (Mody and MacDonald, 1995). increase in [Ca ]j is directly from 2+  [Ca ]i 2+  2 +  release from intracellular  It is not known what proportion of the  entry through NMDA ion channels, and how  much is derived from other sources, such as voltage-operated calcium channels (Tsien et al., 1988) activated upon glutamate-induced membrane depolarization (Dingledine, 1983), non-specific membrane "leak" due to compromised integrity of the plasma membrane (Choi, 1988b), and [Ca *],- or other messenger- induced release from 2  intracellular organelles such as the endoplasmic reticulum (Mody and MacDonald, 1995). What is known from the delayed phase block by A P V (Choi et al., 1988) is that the [Ca ] influx via NMDA receptor-coupled ion channels is a critical initial step in 2+  (  signalling the changes that the neuron undergoes during excitotoxicity. 1.3.  Pharmacology of excitotoxicity  Interventions anywhere along the chain of events occurring under excitotoxic conditions would be expected to protect neurons from excitotoxicity-related damage.  -19The pharmacology of NMDA receptors is particularly well described and hence at this stage is a reasonable initial approach in the management of glutamate-induced C N S injury. Experiments in vivo involving conditions of hypoxia, ischEmia, or hypoglycaemia have demonstrated the efficacy of NMDA antagonists in attenuating the C N S damage observed in such situations (Foster et al., 1988; Gill etal., 1988; McDonald etal., 1989; Kochhar et al., 1991; Lipped et al., 1994). Excitotoxicity experiments using high levels of NMDA or glutamate to induce damage in cultured neuronal preparations have verified the efficacy of NMDA antagonist drugs in protecting neurons from excitotoxic injury (Rothman et al., 1987; Goldberg et al., 1988; Choi ef al., 1988; Tortella et al., 1994) and suggest that this type of experiment may serve as a convenient screen for neuroprotectant compounds. 1.3. J. 1. The concept of "delayed rescue "  For optimal treatment of excitotoxicity-related events, an intervening agent would have to be present during the time of the injurious episode. If a set of conditions or a previous incident is present that would produce a raised probability of the occurrence of an excitotoxicity-related episode, such as a previous stroke, then prophylactic treatment, if appropriate (but see Section 1.4.1.2), could be applied. However, many situations, such as head trauma or a first stroke, strike suddenly and without warning. In these cases, the most beneficial type of therapy could be effective even if applied sometime after the injury. There is a substantial amount of evidence to indicate that the chain of events that results in excitoxicity-induced neuronal necrosis is a timedependent phenomenon (e.g., see Choi, 1987a; Choi et al., 1988) and that there is a  - 20"therapeutic window" during which treatment is expected to be successful.  The  "delayed rescue" of CNS neurons, both in vitro and in vivo, has been demonstrated in a variety of situations (Rothman et al., 1987; Steinberg et al., 1988; Gill et al., 1988; Hartley and Choi, 1989; Bakker and Foster, 1991; Kochhar et al., 1991).  The  antagonism of EAA-induced toxicity is therefore a rational form of therapy for stroke and CNS trauma, as it affords the clinician a certain amount of time to apply treatment before irreversible damage occurs.  1.3.2. Uncompetitive NMDA antagonists One class of NMDA inhibitors, the uncompetitive antagonists, acts by decreasing current flow through the NMDA ion channel subsequent to receptor activation. Compounds from this class, including MK-801 (Wong et al., 1986) and the dissociative anaesthetic ketamine (Martin and Lodge, 1985; MacDonald et ai, 1987), bind to the P C P (phencyclidine) site within the channel pore (Collingridge and Lester, 1989; MacDonald et ai, 1991). The ability of uncompetitive NMDA antagonists to protect neurons from excitotoxic challenge has been well-documented (Weiss et al., 1986; Lysko et al., 1989; Hartley and Choi, 1989; Grigg and Anderson, 1990; Lippert et al., 1994).  Channel blockers acting uncompetitively may offer some advantage over  competitive NMDA antagonists. For example, due to the nature of uncompetitive block, drug actions on open channels are enhanced during excessive NMDA activation; thus, the effects of uncompetitive blockade, unlike competitive antagonism, would not be overcome by increased levels of agonist (Lipton, 1993; Baukrowitz and Yellen, 1996). At present, however, the clinical utility of uncompetitive drugs has been limited by their  -21neurobehavioural side effects, including motor impairment (Willetts et al., 1990; Rogawski etal., 1991). 1.3.2. J. Opiates  Agents from the opiate class of drugs have been found to protect neurons against challenge by high levels of NMDA (Choi and Viseskul, 1988). This protective effect occurred despite an NMDA ion channel-stimulating effect induced by opiatemediated stimulation of intracellular P K C activity (Chen and Huang, 1991; see section 1.1.3.10).  Opiates that protected neurons from NMDA-induced injury included  methadone, morphine, dextrorphan, and levorphanol; others that did so but much more weakly (i.e. with higher ICso's) were fentanyl, dextropropoxyphene, naloxone, and nalorphine. The high concentrations employed (millimolar or high micromolar range), the reversed stereoselectivity compared with classical opioid effects, and the effectiveness of the opioid antagonists naloxone and nalorphine in this model suggest that the protective effects did not occur through classical u, 6, or K receptors. On the other hand, the K-opiates U50,488H and U62,066E (spiradoline mesylate) protected hippocampal neurons in an in vivo gerbil ischaemic model while the close structural relative but non-K compound U54494A did not have any effect (Hall and Pazara, 1988; Gomez-Pinilla et al., 1989).  It is unknown whether or not the endogenous K-opioid  dynorphin A exerts its modulatory effects on NMDA receptor activity (Shukla and Lemaire, 1994) through mechanisms similar to those through which opiates produce anti-excitotoxic neuroprotection, although it should be noted that the selective K-opiate  - 22ethylketocyclazocine has been shown to have no effect on NMDA-evoked responses in cat spinal cord (Berry et al., 1984).  1.3.2.2.Antitussives  One class of drugs that has shown considerable promise for clinical management of excessive NMDA activity, such as might occur in stroke or epilepsy, is the antitussives. Many of these agents were originally thought to behave as opiates in their  antitussive actions, similar to the u-opioid  receptor-mediated  depressive effects produced by compounds from the opiate class.  respiratory  Indeed, many  antitussives, such as the morphinan dextrorphan and its derivative dextromethorphan, are simply optical enantiomers of classical opiates, levorphanol and methyllevorphanol, respectively.  However, since many compounds that behave similarly in their  antitussive effects, e.g. caramiphen and carbetapentane, are not related to opiates, it is now accepted that this class of compounds exerts its antitussive effect through a C N S mechanism distinct from the opioid system.  Another important feature of DM's  pharmacological profile was demonstrated by the discovery of a distinct high-affinity dextromethorphan (DM) binding site in the brain (Craviso and Musacchio, 1983a,b). Non-opiate antitussives have been shown to reduce NMDA responses (Fletcher et al., 1995). The stereoselectivity for NMDA antagonist action (dextrorphan > levorphanol) is the opposite to that seen with opioid effects (levorphanol > dextrorphan) (Church et al., 1985). Binding data have shown dextrorphan to compete for [ H]PCP binding in rat 3  brain cortex and to share a binding locus with many other P C P site ligands (Mendelsohn et al., 1984).  The observation of selective NMDA blockade by  -23dextromethorphan and dextrorphan, with levorphanol being considerably weaker, in cat and rat spinal neurons confirmed their putative actions on the P C P site in or near the NMDA ion channel (Church et al., 1991b).  A neuroprotective effect against NMDA  challenge that parallelled the results of Church et al. (1991b) has been demonstrated (Choi, 1987b). It should be noted that all of the above effects occur at concentrations somewhat (~10X) above those that are believed to occur in the CNS for remediation of cough, suggesting the antitussive and neuroprotective effects are mediated by separate systems. The demonstration of DM's neuroprotective efficacy in vivo have led to recent clinical trials for dextromethorphan and dextrorphan as adjuncts in stroke therapy.  1.3.2.3. Sigma ligands  Many agents that bind to the P C P site in the NMDA ion channel are also ligands of the sigma (a) receptor, an entity once thought to be a subtype of the opioid receptor but now considered to be separate from the other opioid receptor subtypes, p, 5, and K. For a review of a receptor pharmacology and function, see Walker et al. (1990). Sigma agents have been found to modulate the NMDA receptor-ion channel complex in a biphasic manner, with low (nanomolar) concentrations potentiating NMDA activity (Monnet et ai,  1990; Monnet et ai,  1992) and high (micromolar) concentrations  inhibiting NMDA activity (Keana et ai, 1989; Monnet et ai, 1992; Fletcher et ai, 1993). While it is possible that the depression of NMDA receptor-mediated events by a ligands represents a direct action of these agents on NMDA ion channels, little information regarding this issue is available.  - 24-  1.3.3. (-)-B-cyclazocine Inhibition  of  NMDA  responses  by  5,9-dialkylbenzomorphan  "analgetic  antagonists" (Gordon et al., 1961) such as pentazocine and a-cyclazocine shows many of the properties expected of uncompetitive channel blockers acting at the P C P site (Thomson and Lodge, 1985; Sagratella et al., 1985; Church and Lodge, 1990); compounds from this class also serve as neuroprotectants (Olney et al., 1986; Lysko et al., 1992). The related compound (-)-p-cyclazocine (the trans isomer of a-cyclazocine; Lasagna et al., 1964) accesses the C N S (Lasagna et al., 1964) and has been found to interact with the P C P site of the NMDA channel (Todd et al., 1990). In a rat cortical wedge preparation, (-)-p-cyclazocine inhibited depolarizations induced by NMDA; in rat spinal cord neurons studied in vivo, the agent attenuated the increase in firing rate produced by iontophoretic application of NMDA (Church et al., -1991a).  Both of the  above experiments, employing extracellular macroscopic recording techniques, showed that (-)-p-cyclazocine has an range.  IC50  against NMDA responses in the submicromolar  The high potency of (-)-p-cyclazocine suggests detailed studies with this  compound to elucidate its molecular mechanism of action are warranted. Furthermore, the NMDA antagonist properties of this agent earmark it as a candidate for neuroprotection; therefore, assessment of (-)-p-cyclazocine's ability to protect neurons from excitotoxic challenge would have the utility both of providing information about a novel neuroprotective compound and of demonstrating the predictive power of NMDA antagonism data for screening potential neuroprotectants.  - 25-  1.3.4. Dextromethorphan As discussed in section 1.3.1.2, dextromethorphan (the in vivo effects of dextromethorphan are no different from those of dextrorphan: methylesterifying the free hydroxyl group of dextrorphan produces dextromethorphan, and this process is reversed in vivo to the extent that much of the former is available to the site of action; Tortella et al., 1994) has been shown to protect neurons from NMDA challenge (Choi, 1987b), to produce neuroprotection in animal ischaemic models (Steinberg et al., 1988; George et al., 1988), and is currently undergoing clinical trials for use against strokeinduced brain damage.  While its neuroprotective property (Choi, 1987b) may be  related to its action against NMDA responses (Church etal., 1985), little is known about its interaction with single NMDA receptor-ion channels. The assumption that it acts uncompetitively to block NMDA-induced currents by blocking open NMDA ion channels has never been tested; this information is crucial to understanding how blockade of NMDA ion channels proceeds towards neuroprotection, and might aid in the development of dextromethorphan-related agents that would be useful in clinical therapy.  1.3.5. L-687,384 L-687,384  (1-benzylspiro[1,2,3,4-tetrahydronaphthalene-1,4-piperidine])  is  a  high affinity ligand for haloperidol- and ditolylguanidine (DTG)-sensitive sigma sites. In an in vitro radioligand binding assay, [ H]DTG was displaced by L-687,384 from crude 3  guinea pig brain membranes; another a ligand, [ H]SKF-10,047, was potently displaced 3  by L-687,384 in an in vivo radioligand binding assay (Middlemiss et al., 1991). In this study, L-687,384 had minimal affinity for D2 dopamine receptors, a-adrenoceptors, and  - 265-HTIA serotonin receptors.  In vitro displacement of [ H]haloperidol by L-687,384 from 3  rat and human cerebellum was subsequently demonstrated (Barnes et al., 1992); in these authors' hands, L-687,384 was the most potent and selective agent for a receptors compared to a number (> 20) of other a agents. Like other a ligands, L687,384 produced a bell-shaped concentration-response curve when tested on NMDAevoked excitations in vivo, showing NMDA antagonism at high concentrations (Bergeron etal., 1992). 1.4.  Experimental approach  14.1 Rationale For an agent to be appropriate for the clinical management for conditions of hypoxia and ischaemia, such as in thrombotic or aneurytic stroke, hypoglycaemia, or CNS trauma, certain conditions must be met. For example, the agent in question must be stable enough to survive in storage, and to survive the route of administration; the body's metabolic processes must allow the agent to be present in sufficient quantities to be effective at the site(s) of action. Other considerations are outlined in the sections below.  1.4.1.1. Blood-brain barrier  To be useful as a C N S agent in clinical practice, a systemically administered compound must be sufficiently lipophilic to traverse the blood-brain barrier.  Many  compounds can be shown to be effective as neuroprotectants in in vitro models but would not offer any utility as clinical agents because of low blood-brain barrier permeation. This is illustrated by the competitive antagonist A P V , which can protect  -27neurons from the damaging effects of anoxia in vitro {e.g., Grigg and Anderson, 1990) but whose molecules contain sufficient charge that blood-brain barrier penetration is negligible.  1.4.1.2.Lack of toxicity  A suitable agent would be minimally toxic, both within and outside the CNS. A striking example of the toxicity problem arises in the case of the anticonvulsant uncompetitive NMDA antagonist MK-801, which is an effective neuroprotectant and anticonvulsant in vitro (Wong et al., 1986; Huettner and Bean, 1988; Lippert ef a/., 1994), yet produces far too many untoward C N S effects, such as motor impairment, to be useful in the clinic (Willetts et al., 1990). The toxicity of MK-801 has been attributed to its length of action, which in turn is manifested by its prolonged interaction with its binding site in the NMDA ion channel (Chen et al., 1992; Lipton, 1993). Analysis of the kinetic parameters associated with the drug-channel site interaction revealed slow rates for both association with and dissociation from the channel site (Chen et al., 1992), whereas better tolerated compounds exhibited faster kinetics of interaction. The identification of compounds that interact with the binding site quickly, rather than slowly, would be a powerful tool in screening for effective yet safe neuroprotective agents (Rogawski, 1993; Lipton, 1993). Therefore, when assessing the suitability of a given agent as a neuroprotectant, kinetic parameters are of great initial importance.  1.4.1.3.Selectivity  An agent is most useful if it affects only the specific process(es) involved in the pathology in question. In the present case, the central importance of NMDA receptors  - 28in mediating the effects of excitotoxicity makes it a rational target for the amelioration of the damaging effects of excitotoxicity. Activity of an otherwise suitable compound against other, non-excitotoxicity-related, systems would be undesirable and could contribute significantly to unwanted side effects.  Therefore, studies of a potential  neuroprotectant's selectivity for the NMDA responses, compared with responses mediated by other systems, such as non-NMDA receptors and voltage-gated ion channels, would be a crucial step in assessing its clinical suitability. 1.4.1.4.Functional efficacy  The final step in assessing a candidate drug's therapeutic potential as a neuroprotectant in vitro is to apply the drug to whole neurons that have been challenged with excitotoxic conditions. NMDA, at a concentration of 1 mM, is sufficient to induce delayed-phase neurotoxicity in vitro and is roughly analogous to conditions present during in vivo excitotoxic stress.  The potency and efficacy of a candidate  neuroprotectant can then be studied by exposing the stressed cells to varying concentrations of the blocking agent and assessing its effect on the NMDA-induced damage. 1.4.2. Hypothesis Desirable characteristics of neuroprotectant drugs can be predicted by analysis of a given agent's kinetic parameters of interaction with the NMDA receptor-ion-channel complex, as well as that agent's selectivity for NMDA responses.  - 29-  1.4.3. Tissues employed J. 4.3. J. Cultured neurons  Cultured neurons from the rat were chosen for study for a number of reasons. A great deal of work has been done on this type of neuron, in this laboratory as well as other laboratories, and their characteristics are well-described. Prior demonstration of NMDA receptor-ion channel activity indicates that these neurons are suitable for studies in which NMDA activity is a necessary component. The ease of manipulation of cultured neurons allows a variety of treatments to be performed on the same type of preparation; the neurons can be maintained in culture for up to 3 weeks and cells can be available at any given time for experimental use. Lastly, the large number of cells available from cultures greatly facilitates experiments in which large quantities of cells are required.  1.4.3.2. CNS areas used  Both hippocampal and cortical neurons were selected for use. Different areas of the  C N S are differentially  sensitive to excitotoxic  damage (Baumgarten  and  Zimmerman, 1992); therefore, it was necessary to study 2 cell types so that the results would be applicable to a wider range of CNS cell types than if only one type were used. The characteristics of neurons from the hippocampus and cortex are well-described (Mattson and Mark, 1996) and the use of cells from these two areas would facilitate comparisons with previously published data. Pyramidal neurons of the hippocampus in vivo are exquisitely sensitive to hypoxic/ischaemic damage (Kirino, 1982; Suzuki et al., 1983). While it can be argued that the hippocampus may be in a position to be the  -30most affected by an interruption in blood flow, as in hypoxia-induced vasospasm, the observation that granule cells in the dentate gyrus of the hippocampus are strikingly resistant to this treatment suggests pyramidal neurons possess a susceptibility to excitotoxic damage not necessarily shared by all central neurons (Smith et al., 1984; Siesjo, 1986).  Neurons of the frontal cortex also show vulnerability to ischaemic  damage, but less so than hippocampal neurons (Pulsinelli et ai, 1982; Pulsinelli and Duffy, 1983; Smith et ai, 1984; Siesjo, 1986). Nevertheless, neocortical neurons are important because of their proclivity to excitotoxic damage following epileptic seizures or certain types of stroke. Additionally, conditions which involve degeneration of frontal cortex neurons, such as Alzheimer's disease, may involve excitotoxic mechanisms, and it is therefore useful to examine the properties of this cell type that are relevant to excitotoxicity and the amelioration of this process (Baumgarten and Zimmerman, 1992). A series of experiments were carried out in neurons to ascertain effects of DM and (-)-p-cyclazocine on voltage-gated ion currents. The purpose of the experiments was to determine if the compounds could have significant effects to alter electrical transmission in neurons. In particular, interactions of these compounds with voltagegated currents involved in action potential generation, such as sodium (l ) Na  and  potassium (l ) currents, might compromise their use as neuroprotectants. The cultured K  neurons used in the NMDA experiments (either hippocampal or cortical) were not suitable for these studies since their relatively small size (-10 x ~4 pm) yielded small macroscopic currents.  Thus, although l  Na  and l were visible, they were sufficiently K  small to preclude pharmacological analysis. Instead, a cell line from mouse dorsal root ganglion developed by Dr. S. U. Kim has been employed in the macroscopic current  -31experiments.  The cells from this line were larger than either the hippocampal or  cortical neurons, with lengths up to 20 um, and were found to express robust l  N a  and l . K  1.4.3.3. Cardiac myocytes Care must be taken in the clinical application of agents that affect ion channel function because they may not affect only neuronal tissue, but non-neuronal excitable tissue as well. The heart is an example of an organ consisting of the latter type of tissue, and particular attention must be paid to an agent's possible toxic effects on cardiac ion currents if it is to be tried for clinical practice, for example. Therefore, cardiac ventricular myocytes acutely isolated from adult Wistar rats were also employed in this series of experiments in order to check for side effects of (-)-(3cyclazocine and DM on cardiac function. These cells expressed robust l types of K* current:  N a  and two  l (the transient outward K* current) and the delayed-rectifier t o  current. It should be noted that the l l  N a  N a  in myocytes has some different properties from  in neurons; for example, the concentration of TTX required for block of l  myocytes is much higher, by at least 10-fold, than that required to block l  N a  N a  in  in neurons.  The lto is the primary repolarizing current in ventricular myocytes and has similar pharmacology to l in neurons, an example being the block of both currents by 4A  aminopyridine (4-AP).  The delayed rectifier l has similar properties in heart and in K  neurons, as exemplified by the inhibition of both currents by tetraethylammonium (TEA).  - 32-  1.4.4. Strategy A multi-tiered experimental strategy has been utilised in order to provide a rational approach for identifying candidate neuroprotectant drugs. Agents were initially screened for  NMDA antagonist activity  hippocampal and cortical neurons.  using outside-out  patches from both  Selectivity of these agents for NMDA vs. non-  NMDA responses and NMDA ion channels vs. voltage-gated calcium channels was then investigated using microspectrofluorimetric measurements of intracellular calcium responses to excitants specific for the above systems. Use-dependence of the agents was also determined with this technique, thus yielding information about the mechanism of neuroprotectant action. Functional assays on whole hippocampal and cortical neurons in culture were then performed as a final gauge of neuroprotectant activity.  Finally, additional experiments were performed to further elucidate the  biophysical mechanism of action and the selectivity of candidate neuroprotectants, the former involving temperature manipulation of outside-out patches exposed to NMDA and/or putative  neuroprotective drugs, and the latter consisting of whole-cell  experiments measuring effects of the drugs on sodium and potassium currents, using acutely isolated rat cardiac myocytes.  - 332.  MATERIALS AND METHODS 2.1.  Tissue culture  Day-18 Wistar rat hippocampal or cortical cultures were prepared according to the method of Banker and Cowan (1977).  Hippocampi or cortices of day-18 foetal  Wistar rats were dissected, cells were isolated by trituration of the tissue through Pasteur pipettes, and suspended in separate Hank's Balanced Salt Solution (HBSS; Gibco), modified with (mM; source) N a H C 0  3  (4.5; Fisher), glucose (27.8; Sigma),  hydroxyethylpiperazine ethanesulphonate (HEPES) (15; Sigma), penicillin (100 U/ml; Gibco), streptomycin (100 ug/ml; Gibco), and adjusted to pH 7.14 - 7.18 with NaOH (Fisher), for counting in a hEmocytometer.  Once counted, the cell suspension was  diluted to 10 cells/ml, with Dulbecco's Modified Eagle's Medium (DMEM; Gibco), to s  which was added (final concentration in mM) N a H C 0 (45), glucose (8.33), HEPES 3  buffer (10), penicillin (100 U/ml), streptomycin (100 ug/ml), horse serum (20%; Gibco), and, if prevention of yeast contamination was necessary, fungizone (Gibco; if added, this would result in 135 nM amphotericin B and 247 nM sodium desoxycholate in the DMEM) and plated onto 18 mm circular glass coverslips, or, in the case of hippocampal neurons destined for the neuroprotection experiments, onto  poly-D-lysine/laminin  (Sigma)-coated 12 mm circular glass coverslips from a suspension of 3 x 10 cells/ml. 5  Coverslips containing neurons were inserted into Falcon 6-well plates (Becton Dickinson) and incubated in 2 ml modified DMEM (as above) in a 5% C 0 / 9 5 % air 2  atmosphere at 35.5 - 36.0°C until use. Hippocampal neurons were placed into the wells upside-down (i.e., cells on the underside of the coverslip as in Brewer and  - 34Cotman, 1989) while cortical neurons were inserted face-up.  For cortical neurons  intended for use in the neuroprotection experiments, cells were plated directly onto the floor of 12-well tissue culture plates (Falcon) and incubated in modified DMEM. Cells survived under these conditions for up to 8 weeks. In these neurons, [Ca ]j and unitary 2+  current responses to NMDA are fully expressed one week post-isolation and remain stable throughout the second week; cells that had been between 7 and 17 days in culture  were  experiments.  used  for  the  electrophysiological  and  microspectrofluorimetric  In the neuroprotection experiments, cells were used 1 3 - 1 4 days  following isolation, as this age showed maximal susceptibility to the degenerative effects of NMDA.  2.2.  Electrophysiology  The advent of patch clamp techniques for measurement of cell membrane currents has allowed investigators to study the behaviour of single ion channels. Additionally, using the patch clamp technique, current through the entire cell membrane can be measured with minimal disruption of the membrane, thus averting leakage of cell contents into the extracellular medium and allowing the cell to maintain its own membrane potential.  This compares favourably with conventional intracellular  microelectrode recording where the cell is punctured, resulting in leakage of cellular contents into the extracellular medium and loss of membrane potential. The ability of the patch clamp technique to detect tiny currents in cell membranes is due to two advances that occurred in the field of electrophysiological recording in the late 1970's, the first being the inclusion of a high resistance feedback resistor in the recording headstage, and the second being the development of a procedure that allowed the  - 35formation of a high-resistance, gigohm seal between the membrane and a solutionfilled glass recording electrode (Hamill et al., 1981) so that current leak between the electrode and the membrane patch was eliminated.  2.2.1. Patch clamp configurations The details of patch-clamp recording are too extensive to completely describe in this thesis; for a thorough treatment of the technique, consult Hamill et al. (1981) and Sakmann and Neher (1984). Briefly, there are four configurations that one can use to record membrane activity.  The first, and most basic,  is the "cell-attached"  configuration, in which unitary currents are measured while the recording electrode is positioned on an intact cell. Excising the patch by pulling the electrode away from the cell produces a membrane patch on the tip of the electrode in the "inside-out" configuration. In this mode, the inner surface of the membrane patch is exposed to the contents of the external recording medium.  Alternatively, once the cell-attached  configuration is achieved, the patch of membrane enclosed by the recording pipette can be broken, using either high suction or high-voltage pulses.  This makes the  interior of the cell continuous with the contents of the pipette so that currents through the entire cell membrane can be detected; this is the "whole-cell" mode of recording and was used in the present work to study macroscopic currents in cultured mouse DRG neurons and isolated rat ventricular myocytes. The final configuration involves removing the electrode from the cell, as is done to achieve the inside-out configuration. However,  if this  manceuver is performed while  starting  from the whole-cell  configuration, instead of the cell-attached mode, the patch spontaneously forms the "outside-out" configuration.  - 362.2.1.1. Outside-out patches  The outside-out patch configuration is the most informative single-channel mode in terms of investigating drug potency and mechanisms.  In this preparation, the  exterior surface of the membrane is exposed to the extracellular medium, while the contents of the recording electrode replace the internal environment of the cell. One is thus enabled, through exchange of the surrounding bath medium, to apply several treatments, such as several drug concentrations, along with control and recovery, to a single patch. A drawback to the outside-out configuration is that it is the most difficult patch clamp preparation to achieve and maintain. It is necessary to attempt many trials before a reasonably stable and noise-free patch can be obtained. As well, the period when the patch- signal is "clean" enough to discern single-channel activity is often too brief to perform an entire experiment; indeed, many patches last-only seconds before the patch seal deteriorates to an unacceptable level.  Furthermore, a patch that is  suitable for study ideally contains only one active ion channel. Many patches exhibit no channel activity. Others have more than one active channel; the records from these patches are too complex to interpret and thus, as with silent patches, must be discarded. As for the presence of other active ion channel species in a patch, these must be suppressed either by pharmacological means or by using an ionic environment appropriate to the system being studied.  For example, when studying NMDA ion  channels (which are permeant to Na* and K*), voltage-dependent Na* and K* channel activity must be inhibited by the addition of tetrodotoxin to the bath and by the use of caesium to replace potassium in both the bath and the pipette.  Another potential  drawback is the extent to which the manipulations performed to achieve the outside-out  -37configuration disturb the normal functioning of the ion channels in the patch. To this end, work in our laboratory has shown the unitary properties of NMDA ion channels in outside-out patches are comparable to those observed in cell-attached patches, despite loss of intracellular factors upon excision to the outside-out configuration.  2.2.2. Single-channel patch clamp procedure 2.2.2.1.Pipette preparation Patch damp electrodes were fabricated from pipette glass (A-M Systems) made from Corning #7052 glass without microfilaments, with an outer diameter of either 1.2 mm (0.68 mm inner diameter), or 1.65 mm (inner diameter of 1.2 mm). The pipettes were prepared with a standard two-pull technique using a Narishige PP-83 glass microelectrode puller, producing pipette electrodes with tip diameters of approximately 1-2 pm corresponding to a resistance of 4 - 8 M Q once filled with bath solution (see below). The pulled pipettes were then fire-polished on a microforge consisting of a glass-coated heating filament, whose temperature and heating area were controlled by a jet of air, positioned on the stage of an ordinary laboratory ocular microscope. The polished electrodes were filled with pipette solution, consisting of (mM; all from Sigma) CsCH S0 3  3  (140), CaCI  2  (0.5), ethylene glycol-bis(p-aminoethyl  etherJ-N.N.N'.N'-  tetraacetic acid (EGTA; 5) and HEPES (10), immediately before use.  Previous to  filling, all pipette solutions were filtered twice using Millipore Type G S circle filters with a 0.22 pm pore size.  - 382.2.2.2. The recording bath  Coverslips containing neurons were placed in a 5 ml Perspex circular recording chamber to which was added, to a level just above the top of the coverslip, bath solution consisting of (mM; source): NaCI (140; BDH), CsCI (5; BDH), CaCI  2  (1.8;  Sigma), HEPES (10; Sigma), and TTX (0.001; Sigma); the pH was adjusted to 7.2 with NaOH (Fisher). 2.2.1.1.  Caesium was used in place of potassium as described in section  No Mg * was added to any of the solutions, as this divalent cation has 2  profound effects on the kinetics of the NMDA channel (Jahr and Stevens, 1990; Mayer ef a/., 1984; Nowak et al., 1984), but the possibility of contaminating Mg * being present 2  in the solutions necessitated an analysis of these solutions for residual magnesium; the Mg * level in the bath solution, as determined by flame analysis on a Jarrell-Ash 280 2  atomic absorption spectrometer, was established to be 0.4±0.2-uM.  The recording  chamber was affixed to the stage of a Nikon T M S inverted microscope with attached phase-contrast optics.  In this system, viable neurons were easily identified by a  characteristic "glow" and the pipette tip could be clearly seen during the patch formation procedure. The bath was exchanged three times with bath solution after the cover slip was inserted so that any culture medium remaining on the coverslip was completely rinsed away. An agar-filled bridge was submerged in the bath and a wire connected to ground was inserted into the bridge so that the recording circuit was complete. Experiments were performed at room temperature (24°C).  - 392.2.2.3. Pipette mounting  Filled pipettes were loaded into the pipette holder which was mounted onto the headstage (Axon model CV-3 with 1/100 gain). These were mounted onto a Newport 360-90 lab jack fitted with a Newport 423 series actuator powered by a Newport model 861 handheld motion controller for manipulation of the pipette position; the microscope and lab jack were placed on a nitrogen-suspended floating table (Newport) so that the cells in the bath were in a vibration-free environment. After mounting into the pipette holder, a slight positive pressure was applied to the pipette to prevent buildup of debris around the tip area, and the pipette was rapidly lowered into the recording bath and manoeuvred into position just above a suitable neuron.  Only smooth, healthy  appearing cells with a bipolar shape and an average length of approximately 10-12 pm along the long axis were chosen for recording. 2.2.2.4. Patch formation  The pipette was slowly lowered into physical contact with the soma of the cell; pipette contact with the cell was viewed through the microscope and was also monitored using a 0.2 mV test pulse with an audio signal being applied during the lowering procedure. Unitary currents were detected with the Axopatch 1B patch clamp amplifier (Axon Instruments, Foster City, CA), which also sent out the test pulses, and the Axopatch output signal was visualized on a Kikusui 5020 20 Mhz digital oscilloscope. When the pipette was touching the cell, a gigohm (>10 GO.) seal was made by applying a small negative pressure through a mouth suction tube which was hooked up to the pipette holder. The large increase in pipette tip resistance associated  -40with seal formation was monitored by the disappearance of the 0.2 mV test pulse on the oscilloscope screen and was confirmed by the audio feedback signal characteristic of a tight seal. Attempts to form a gigohm seal were not always successful, and if only a partial seal was attainable the pipette was discarded.  A greater suction, and, if  necessary, a 0.2 V pulse of between 1 and 10 ms duration, was then applied in order to enter the whole-cell configuration. Following this manoeuvre, the patch was excised to obtain the outside-out configuration.  2.2.2.5.Patch clamp recording, data acquisition, and storage  The patch-clamp data were acquired using an Axopatch DigiData 1200 interface and recorded onto a 40 MHz 486 P C loaded with Microsoft MS-DOS 3.2 and pCLAMP version 6.0.2 (Axon Instruments Inc.).  For backup recording, an Instrutech VR-10  digital data recorder was connected to a Panasonic Omnivision Hi-Fi MTS VHS, model PV-4760-K. Patch-clamp recordings were sampled at 5 kHz and filtered at 2 kHz with a low-pass -3 dB filter.  In some cases, especially when the frequency of channel  openings was low, an Al 2020 event detector was used so that computer recording commenced only when an event had occurred. Data from outside-out patches were recorded  in 10-second segments,  during  which  a hyperpolarizing  potential,  corresponding to a negative pipette potential (V ), from the range -20 to -100 mV and in p  steps of 20 mV, was applied so that data were collected over a range of membrane voltages (as in Antonov and Johnson, 1996). Several recording segments were made at each V , and the sequence of pipette potentials applied for recording was constantly p  varied in order to eliminate any interactive effects with previously applied voltages. No  - 41currents were apparent when patches were held at 0 mV, consistent with a reversal potential of 0 mV for [Na ] = [Cs ]i = 140 mM. +  +  0  2.2.2.6.Perfusion of solutions  Bath solutions, as in section 2.2.1.3, containing agonists (10 pM NMDA and 1 pM glycine; both from Sigma) or agonists + antagonists were stored in inverted syringe tubes suspended above the level of the bath and allowed to flow into the bath by gravity via polyethylene connecting tubes at a rate of approximately 2 ml/min. Solution volume in the bath was kept at just over 2 ml by removing solution by vacuum suction. After forming a stable outside-out patch and any ambient activity recorded, agonist solution consisting of bath solution plus 10 pM NMDA and 1 pM glycine was added to the bath.  Providing unitary currents with characteristics corresponding to those of  NMDA ion channel activity (generally, the most observable criterion was the value for the channel conductance, which for NMDA ion channels in outside-out patches is in the range 40-50 pS; Ascher and Nowak, 1988; Bertolino et ai, 1988) were apparent in the patch, recordings were then made at various patch potentials. After this, the solution in the bath was switched to the same solution with the addition of a putative antagonist. The antagonists studied were dextromethorphan (5 - 50 pM), L-687,384 (5 - 40 uM; a gift from Dr. D. N. Middlemiss, Merck, Sharp, and Dohme Research Laboratories, Harlow, U.K.), (-)-p-cyclazocine (0.05 - 4 pM; Sterling-Winthrop, U.K.), and (+)-0cyclazocine (1 - 10 pM; Sterling-Winthrop, U.K). Generally, for addition of agonist solution, and for addition of agonist + antagonist solution, a total of 10 ml was allowed to flow out of the syringe tube; this was more than sufficient for complete exchange of  -42the bath solution. For recovery conditions, where solution containing only the agonists was used to wash out antagonist, a greater amount of solution was necessary for complete removal of antagonist from the patch; in some cases this required up to 30 ml. 2.2.2.7.Data analysis Unitary currents were analyzed using the Fetchan module from pCLAMP. Using this module, single-channel events in the data records were selected manually according to the criteria outlined below, and the amplitude, open time, and duration of the previous closed interval for each selected event were stored in an idealized record. Events selected for the idealized record were constrained to conform to minimum amplitude criteria (see Section 2.2.2.8); these criteria were unique for each pipette voltage and were established such that selecting events conforming to these criteria would produce monotonic, normally-distributed amplitude histograms.  With the filter  settings used and the seal resistances obtained in this study, only openings of 400 us or greater were properly resolved; openings briefer than this were not selected for the idealized records. The minimum resolvable closed time duration was set at 100 ps. The pSTAT subroutine of pCLAMP was then used to calculate the mean amplitudes, mean open times, mean event frequencies, and closed time constants from this idealized record. This module was also used to generate histograms. The amplitude, open time, and closed time histograms, such as those shown in section 3.2, were plotted using a Hewlett Packard Color Pro graphics printer. A minimum of 200 events was included in each open and closed time histogram for kinetic analysis, except where high concentrations of potent antagonists were applied, necessitating a lower minimum  - 43of 100 events per histogram because of the strong effect of some drugs to lower channel opening frequency. The open and closed time histograms were curve fitted by pSTAT, which uses a non-linear least-squares curve-fitting routine to fit 1 - or 2exponential functions by the Levenberg-Marquardt (LMQ) technique with Simplex optimization.  In this routine, the data were weighted by their % values and, starting 2  from seed values that were either manually entered or automatically determined by pSTAT, the fit proceeded until convergence, defined either when improvements in x* were less than 2.5 x 10' , or if all of the parameters converged when parameter change 7  vectors went to zero. In order to determine if a particular histogram could be fitted with more than two exponentials (as in the case of the closed duration histograms), the two fast time components would first be fit to the left-most portion of the histogram; then, the curve fit would be performed again with the data range now modified to include the portion of the curve fitted with the second component plus the data to be fit with a third component, and so on. Errors are expressed as ±S.E.M., where n refers to the number of patches studied; experiments were performed at room temperature (24°C).  2.2.2.8.Identification of unitary currents through NMDA ion channels A patch may contain ion channels other than the NMDA channel under investigation. The following is a list of criteria used to identify unitary currents carried by NMDA ion channels,  (i) Channel conductance.  The NMDA ion channel is  characterized by a conductance state with a conductance (G) value of 40 - 50 pS (Ascher et al, 1988; Ascher and Nowak, 1988). At a given V , unitary currents with p  amplitudes that did not conform to within 10% of this conductance range were not  - 44included for analysis. It has been suggested that NMDA ion channels may themselves have multiple conductance states (Jahr and Stevens, 1987), with other states having conductance values lower than the primary 40 - 50 pS conductance state.  When  smaller currents were intermittently observed in the present electrophysiological experiments (see Section 3.2.1.1), they were not included in the analysis since the aim of the study was to investigate the primary high-conductance state of the NMDAactivated ion channel, (ii) Requirement for glycine. Unitary currents conforming to the 40 - 50 pS conductance criterion were observed at a vanishingly low frequency (< 0.1 Hz) when NMDA, without glycine, was perfused onto outside-out patches; perfusing NMDA with 1 uM glycine resulted in the activation of unitary currents at frequencies in the expected range (see Sections 3.2.3.4 and 3.2.9.4). Correlation of the frequency of NMDA-activated unitary currents and occupancy of the strychnine-insensitive glycine site has been previously documented (Johnson and Ascher, 1987). (iii) Sensitivity to 2-  amino-5-phosphonovalerate (APV). Currents observed following application of NMDA and glycine to outside-out patches that conformed to the 40 - 50 pS conductance requirement were not observed if 100 uM APV was included in the perfusion solution (n = 4).  Removal of A P V from the perfusion solution resulted in the reappearance of  unitary currents. A P V is a competitive antagonist of NMDA receptors which acts by displacing agonist from its binding site (Peet et al., 1986; Olverman et al., 1988). (iv) Sensitivity to Mg *. Application of Mg * to the external surface of outside-out patches 2  2  exposed to NMDA and glycine produced an alteration in the appearance of unitary currents that was characterized by numerous fast transitions between an open level and a non-conducting level ("fast flickering"; cf. Figure 1). This action of Mg * is well2  -45known and represents fast-kinetics block of open NMDA ion channels (Nowak et al., 1984; Jahr and Stevens, 1990). The single-channel patch clamp studies in the present work are based on the behaviour, and the response of that behaviour, to a set of compounds, of unitary currents fulfilling the above criteria. 2.2.2.9.Statistics As patches tested with the compounds served as their own control (i.e. control and drug values were non-independent), data were generally expressed as the mean of the percents of the value obtained in the presence of the compounds tested to that obtained in the control for each patch, ± the standard error of the mean. Channel parameters (e.g. mean open time, channel open probability) were assumed to be normally distributed and therefore parametric Mests utilizing the ratios were performed as outlined in Howell (1987); parametric statistics were obtained only for values where n > 3 (Antonov and Johnson, 1996). The P values obtained in the tests, representing the probabilities of Type I errors [i.e. the probabilities that the claimed treatment differences could have occurred by chance), have been included in the Results section. 2.3.  Microspectrofluorimetry  2.3.1. Fura-2 experiments A digital fluorescence microscopy system (Attofluor), set up for the dualwavelength excitation ratio method (Grynkiewicz et al., 1986), was used with hippocampal or cortical neurons, prepared as in section 2.1, loaded with fura-2, a fluorescent free-calcium indicator dye (Grynkiewicz et al., 1986) to assess the effects of dextromethorphan,  L-687,384, and (-)- and (+)-R-cyclazocine on NMDA-induced  - 46increases in free intracellular calcium, according to the procedure outlined below. In addition, this technique was used to determine the selectivity of (-)-p-cyclazocine for inhibiting NMDA activity relative to its effects on responses elicited via a-amino-3hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors, kainate receptors, or highvoltage-gated calcium channels. 2.3.1.1 .Fura-2 loading Medium containing the acetomethoxy ester of fura-2 (fura-2-AM) was prepared by dissolving 2 ul stock fura-2-AM (Molecular Probes, Eugene, OR) in 25 ul dimethylsulphoxide (DMSO) and vortexing. Stock fura-2-AM was previously mixed with the dispersing agent Pluronic F-127 (Molecular Probes; see Poenie et al., 1986) before storing at -70°C and was thawed immediately before DMSO addition. This solution was slowly added, while vortexing, to 3 ml of loading medium, consisting of (mM) NaCI (136.5), KCI (3.0), N a H P 0 (1.5), glucose (10), H E P E S (10) with pH adjusted to 7.40 2  4  with NaOH, to which was added 10 ul bovine serum albumin (BSA; thawed by vortexing from stock previously frozen at -70°C). Each 18 mm cover slip containing foetal rat hippocampal or cortical neurons at a density of 10 cm' was loaded, using forceps 5  2  sterilized in 75% ethanol, into 1 ml of this medium + 1 ml loading medium, without fura2AM/DMSO/BSA, for a final fura-2 concentration of 7.1 uM, for 80 min at 35°C in 5% C0 /95% air and then washed by incubating at room temperature (20°C) in 3 ml loading 2  medium, without fura-2AM/DMSO/BSA, for at least 30 minutes. This last wash step was used to ensure complete hydrolysis of the esterified dye so that leakage of fura-2  -47both from the cells to the extracellular medium and into intracellular organelles was minimized (Puil et al., 1990). 2.3.1.2.Measurement offura-2 fluorescence  A coverslip containing fura-2-loaded ceils was inserted into a Perspex laminar flow-through chamber which in turn was inserted into a stainless steel bath; a rubber ring lined with silicone gel was used to seal the coverslip into the holder and prevent leakage of solution into the spectrofluorimetric apparatus. The chamber was locked into position on the stage of a Zeiss inverted microscope with phase-contrast optics and equipped with an imaging camera. A peristaltic pump was used to deliver solution from holding reservoirs into the bath chamber, at 2 - 3 ml/min, via an inlet port and suction was used to keep the solution level just above the top of the cells. The preparation was perfused with the same loading medium used above (without fura-2AM/DMSO/BSA) to which 0.3 pM TTX and 2 pM glycine had been added. Rises in [Ca ]i were evoked by 20 s bolus applications of loading medium to which either 20 pM 2+  NMDA, 80 pM kainate, 40 pM (±)-a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), or 50 mM K* (by substitution for NaCl) were added. Dextromethorphan, ( 5 - 1 0 0 pM), L-687,384 (10 - 100 \M), (-)-p-cyclazocine (0.02 - 5 pM), or (+)-pcyclazocine (5 pM) were added through superfusion. A DC-powered 200 W Hg arc lamp was used to illuminate the cells for [Ca ]j measurement; a stepmotor-driven turret 2+  operated by computer was used to switch between two 10 nm bandpass differential interference filters to give first 334 nm and then 380 nm light for determination of bound [Ca *]j and free [Ca *]j, respectively. The ratio of these two intensities gives a measure 2  2  -48of [Ca ]i which is independent of fura-2 concentration (Grynkiewicz et al., 1985). 2+  During measurement of responses to excitant applications, ratios were acquired every 2 s; this rate was decreased to one every 20 s between excitant applications in order to minimize photobleaching and ultraviolet light-mediated cytotoxicity.  Fluorescence  intensities were detected at 510 nm from multiple neuronal somata simultaneously. The fluorescence signals were corrected for background before calculation of the ratio by measuring fluorescence intensity emitted from a portion of the coverslip where no cells were present. All fura-2 measurements were taken at 20°C.  2.3.1.S.Cdlculation  offCcf+Jt  The concentration of cytosolic free calcium was calculated from the ratio of the emitted fluorescence at 334 nm to 380 nm (i.e., bound to free calcium) according tp the method of Grynkiewicz et al, (1985). Briefly, [Ca ]i = Kd x p x (R - R n)/(Rmax - R), 2+  mi  where Kd is the equilibrium dissociation constant for the association of fura-2 with cytosolic free calcium, p is the ratio of 380 nm light with zero [Ca ]j, to 380 nm light 2+  with infinite [Ca ]i, R is the experimentally determined ratio of 334 nm to 380 nm light, 2+  Rmin is the ratio of 334 nm to 380 nm light with zero external calcium ([Ca *]j), and Rmax 2  is the ratio of 334 nm to 380 nm light with infinite external calcium. The values for p, R , and Rmax were determined from calibration procedures using 10 uM of the calcium min  ionophore Br-A23187 either in the absence of [Ca ]j, with 100 uM EGTA, or with 2+  excess (2 mM) [Ca ]j. 2+  The published Kd value for fura-2 of 135 nM (at 20°C) was  employed (Grynkiewicz et al., 1985).  Responses were measured as the difference  between [Ca *]i at the peak of the response and [Ca ]i at the baseline immediately 2  2+  - 49previous to the excitant challenge. In this way drift of the baseline is accounted for. Results are expressed as mean±S.E.M., where n refers to the total number of neurons from which data were obtained under each experimental condition. Each experiment was performed on at least three different neuronal cultures. 2.4. The  Neuroprotection experiments properties  of  (-)-  and (+)-p-cyclazocine  as neuroprotectants  were  investigated by assessment of cell viability with the application of high concentrations of NMDA to cultured hippocampal or cortical neurons. Viability was assessed using a quantitative  morphometric technique described below.  Neurons were kept  in  maintenance medium (DMEM (Gibco) plus (mM; source), N a H C 0 (22; Sigma), HEPES 3  (10; Sigma), 10% heat-inactivated horse serum (Gibco), and 6.0 g/L glucose (Sigma); pH was adjusted to 7.40 with NaOH (Fisher). Cultures were prepared in exactly the same manner as for the electrophysiological and microspectrofluorimetric experiments, with the exception of the modifications mentioned in section 2.1, and maintained in a 37°C incubator with a 5%C0 /95% air atmosphere prior to use. All manipulations were 2  performed in a laminar-flow fume hood with all surfaces sterilized by wiping with 95% ethanol. Solutions were added to and removed from the culture wells containing the coverslips by a pipet-aid automatic pipettor (Drummond); all media were filter-sterilized using a Falcon 7107 0.22 Micron Easy Flow filter (Becton Dickinson) and pH-adjusted to 7.40 with NaOH (Fisher) or HCI (Fisher) immediately before use.  (-)- or  (+)-P-  cyclazocine were added using an Eppendorf micropipettor with Eppendorf disposable tips. Initially, the cultures were stained with trypan blue (Sigma; see Paul, 1975), which stains dead cells dark while leaving viable cells unstained, by adding 2 ml/well 0.4%  - 50trypan blue in 0.81% NaCI (BDH) and 0.06% K 2 H P O 4 (Sigma) for 10 minutes, at which time the stain was replaced with maintenance medium.  The neurons were then  counted, using a Nikon TMS microscope, by totalling the number of cells present in seven non-overlapping fields situated at pre-selected locations in the field of cells. Repeated counts of the same culture well confirmed the cell totals to be highly reproducible (within 20%). Only smooth, healthy-appearing cells that were not trypan blue-stained and that conformed to previous descriptions of primary hippocampal neuronal cultures (Olney etal., 1971; Banker and Cowan, 1977; Choi, 1987a; Rothman et al., 1987; Choi et al., 1988; Mattson et al., 1993) were chosen for counting. The culture wells were required to yield at least 100 cells in total when counted in this manner to be considered for later use in the experiment; however, most culture wells used yielded between 300-400 cells by this counting technique. .Ten minutes prior to amino acid excitant challenge, the appropriate amount of (-)- or (+)-p-cyclazocine was added to each of the culture wells, except for those where only NMDA was to be added or those where neither NMDA nor p-cyclazocine were to be added. After 10 minutes, the maintenance medium in the wells was replaced with the same medium except for the addition of 1 uM glycine (Sigma), and, where appropriate, 1 mM NMDA (Sigma) and a given concentration of either (-)- or (+)-p-cyclazocine; all replacement media were adjusted to pH 7.40 with NaOH.  After 24 hours, the cover slips were once again  stained with trypan blue for 10 minutes, as above, and counted in exactly the same manner as before the amino acid challenge. Again, cells that had taken up trypan blue were excluded from the count. Results are expressed as the percentage of neurons remaining viable at the 24-hour count relative to those present in the pre-challenge  - 51count; this is termed the survival rate for a given culture well. For results reported as percent neuroprotection, mean survival rates for (-)- and (+)-p-cyclazocine-treated culture wells within a given culture batch were first corrected for procedural cell loss (for example, suction of cells into the pipette during solution exchange), by dividing by the survival rate obtained in cultures where only 1 uM glycine was added, and then normalized to the survival rate obtained for control culture wells where 1 mM NMDA + 1 uM glycine (with no (-)- or (+)-p-cyclazocine) having been added. Results for control cultures (1 mM NMDA + 1 uM glycine) were also corrected for procedural cell loss as assessed by culture wells to which only 1 uM glycine had been added. The number of separate cultures (i.e., those derived from different mother rats) on which the experiment was performed was eight, which is referred to in section 3 as n. Within a given culture, each treatment was assigned to a minimum of 3 cover slips. 2.4. J. 1.Statistics  The counts for the cover slips within a given culture were averaged and the results were expressed as the mean of the n = 8 such values for a given treatment, ± the standard error of the mean. Cell counts were assumed to be normally distributed across treatments and a one-way analysis of variance, followed by a Tukey post hoc test, were employed to determine significant changes in viability. 2.5.  Temperature studies  Outside-out patches exposed to NMDA in the presence and/or absence of antagonist drugs were varied from 24°C to 10°C by a proportional temperature control system.  The bath temperature was detected by Peltier devices, sampled, and  -52deviations away from a previously determined set point were corrected by a thermistor device coupled to the bath setup. The setup used was similar to that described in Chabala et al., 1985. After formation of an outside-out patch, NMDA and glycine were added to the bath and control data collected. The bath containing the patch was then cooled until a previously determined set point, usually 10°C below ambient room temperature, had been reached, whereupon data were again collected. Recordings were also made during the cooling run. When sufficient data had been obtained at the lower temperature, the solution in the bath was replaced with a new solution containing NMDA, glycine, and drug, and recooled to the original set point.  Data were again  collected before allowing the patch to warm up to room temperature; recordings were made during this warming phase, as well as upon reaching room temperature. When possible, control solution containing NMDA and glycine, but no drug, was used to wash out the drug and recovery data were taken. The cooling rate was set so that over a ten-second data recording interval, the temperature changed by no more than 0.4°C. The temperature response of the system was calibrated using a digital thermometer inserted into the bath containing standard bath solution. The dependence of a certain quantity on temperature was expressed as a Q ±S.E.M., which is the ratio of the 10  values for that quantity over a 10°C change in temperature, expressed so that Q >1. It 10  should be noted that Q10 depends on the specific temperature range used for the calculation; therefore, all Q 14.0±0.2°C.  1 0  values refer to measurements taken at 24.0±0.2°C and  -532.6.  Experiments with voltage-gated Na* and K* currents  2.6.1. Preparation and use ofDRG neurons The neurons employed in the whole-cell experiments were from an immortalized cell line derived from rat dorsal root ganglion neurons and were developed in the laboratory of Dr. Seung U. Kim of the Department of Neurology at the University Hospital site of the University of British Columbia.  Further details concerning the  preparation and maintenance of this cell line should be directed to the above laboratory. The procedures employed in recording macroscopic currents from these neurons were identical to those used for the rat ventricular myocytes and are described below (Section 2.6.3).  2.6.2. Isolation of rat ventricular myocytes The rat ventricular myocytes were dissociated and isolated from male rats (Sprague-Dawley) by slight modifications of procedures described previously (Mitra and Morad, 1985; McLarnon and Xu, 1995). Briefly, hearts were removed rapidly from animals anaesthetized with pentobarbitone and were perfused using a constant-flow Langendorff system with a zero-Ca * Tyrode's solution containing (mM; source): NaCI 2  (133.5; BDH), KCI (4; BDH), MgCI (1.2; Sigma), N a H P 0 2  2  4  (1.2; Sigma), T E S (10;  Sigma), and glucose (11; Sigma); the pH was adjusted to 7.4 with NaOH (Fisher). The perfusate was oxygenated and maintained at 37°C. After 4 min, the perfusate was exchanged for another solution containing 0.07% collagenase (Type II, Worthington Biochemical), 25 uM CaCI (Sigma), and 200 mg of BSA (Boehringer Mannheim). After 2  15 to 20 minutes, the Tyrode's solution (containing 25 uM CaCI ) was re-applied with 2  gentle agitation of the digested tissue. The myocytes were stored at room temperature  - 54between intervals of washing with successively increasing concentrations (up to 100 pM). Cells were plated on coverslips precoated with laminin (4 pg per coverslip) and used for electrophysiological study. Light microscopic examination revealed the cells had a mean length of 72±6 pm (n=22) and a mean diameter of 7.8±0.5 pm (n=22).  2.6.3. Whole-cell electrophysiology 2.6.3. J.Macroscopic  recording  Currents were recorded from rat cardiac myocytes using the whole-cell patchclamp configuration. Macroscopic currents, specifically transient outward K* currents (l ), and inward sodium currents (l ), were recorded from isolated rat ventricular t0  Na  myocytes for periods up to 8 hours after plating. The details of the whole-cell patch clamp configuration are given in Section 2.2.1, and the procedure followed to obtain the whole-cell configuration was nearly identical to the procedure to obtain outside-out patches (Section 2.2.2); the exceptions to the procedure are herewith given.  The  micropipettes used were prepared in exactly the same fashion, except that lower resistances (in the range 2 to 4 MQ) were used in order to improve access to the interior of the cell. The solution used to fill the micropipettes was different from that used for outside-out patch recording and consisted of (mM; source): NaCl (10; BDH), KCI (140; BDH), MgCI (1; Sigma), EGTA (10; Sigma), Mg-ATP (5; Sigma), and HEPES 2  (10; Sigma); the pH was adjusted to 7.4 with KOH (Fisher). The bath set-up was also identical to that described in Section 2.2.2, except that, since voltage-activated macroscopic currents were being studied, no agonists (e.g., NMDA) were included in the perfusion tubes.  An Axopatch amplifier (200A, Axon Instruments) was used to  - 55record currents with the low-pass filter set at 1 or 2 kHz; recording of currents began at the establishment of the whole-cell configuration without any attempt to excise a patch of membrane. Capacitative currents and series resistance were compensated by use of analog circuitry and leak subtraction was also used in some experiments; P/N subtraction was not routinely done and when carried out had no effect on macroscopic currents.  The mean value of seal resistance was 36±5 G Q (n=12); with series  resistance compensation set at 80-85%, the mean series resistance was 4.2±1 Mft (n=10) and the mean cell capacitance was 92±6 pF (n=6). The voltage protocols were generated by the CLAMPEX subroutine of pCLAMP (Axon Instruments) and data were recorded on disk for subsequent analysis. Descriptions of the voltage protocols have been included in the Results (Section 3.5.2). All experiments were carried out at room temperature (20-24°C).  2.6.3.2.Data analysis  The data analysis was performed using the CLAMPFIT subroutine of the pCLAMP software (Axon Instruments).  This program was used to fit the inactivation  time-course of l with single exponential functions, with the time-course denoted by x; to  using dual- or multi-exponential functions did not improve the goodness of fit to the data.  Peak current responses for l and l t0  Na  were also measured using CLAMPFIT.  Comparison was made of values obtained in this manner and those recorded in the absence and presence of either (-)-0-cyclazocine or DM, and were expressed as either the mean of the percentages or the ratios of the latter to the former, ± the standard error of the mean.  - 563.  RESULTS 3.1.  Organization  The organization of this part of the thesis is first considered. compounds have been studied in detail.  These were:  Firstly, four  L-687,384, a ligand for the  putative sigma (a) receptor (Middlemiss et al., 1991); dextromethorphan (DM), a nonopioid antitussive; and the two optical isomers of p-cyclazocine (the geometric isomer of the 6,7-benzomorphan a-cyclazbcine). All of these compounds have potential use as neuroprotectants.  Since similar experimental strategies and protocols have been  applied to characterize drug interactions for all four compounds, the results are considered  on  the  basis  of  actions  on  various  electrophysiological,  microspectrofluorescent, and neuroprotective properties rather than entirely separate sections for each of the compounds. This organization allows a summary of overall salient points to be included in sections labelled as Conclusions; details specific to particular compounds are indicated separately.  Thus, the results are organized as  follows: Electrophysiology (A) Drug actions on amplitudes and conductance of unitary NMDA currents (B) Drug actions on kinetics of unitary NMDA currents (C) Data consistency with open channel blockade (D) Analysis of data using open channel block scheme and conclusions (E) Use of biophysical probes for mechanisms of channel block  - 57Microspectrofluorescence (F) Drug actions on NMDA-evoked [Ca *]j responses 2  (G) Drug selectivity for NMDA-evoked [Ca *]j responses vs. responses mediated 2  via non-NMDA receptors and voltage-gated C a * channels 2  Neuroprotection (H) Neuroprotection assays Actions of the compounds on voltage-gated ion channels (I) Studies of drug actions on Na* and K* currents in neurons and cardiac myocytes Conclusions  - 583.2.  Electrophysiology  The results presented in this section place emphasis on (-)-p-cyclazocine, which was found to exhibit the most utility as a putative neuroprotectant. Thus, for clarity, the figures shown in this section will largely use this compound as a demonstration of drug actions on the NMDA ion channel, along with comparisons to its enantiomer, cyclazocine. Figures showing the actions of the other agents, dextromethorphan (DM) and L-687,384, are included either to illustrate particular effects unique to these compounds, or where the particular properties of these compounds afforded analysis superior to that which could be performed with (-)-p-cyclazocine. An example of the latter case was the determination of the voltage-dependence of drug actions with L687,384. This compound did not reduce frequencies of channel openings, unlike the other compounds; hence, a large number of unitary events could be collected over a range of patch potentials from -100 mV to -20 mV (pipette potential, or V ). p  HIPPOCAMPAL NEURONS 3.2.1. Drug actions on amplitudes of unitary NMDA currents and channel conductance in hippocampal neurons 3.2.1.1.Amplitudes of unitary NMDA currents and channel conductance in control solution Figure 1A shows typical single channel NMDA currents recorded from an outside-out patch isolated from a rat hippocampal neuron. The currents were recorded following application of 10 pM NMDA plus 1 pM glycine to the Mg *-free bath solution. 2  A current-voltage (IA/) plot for the same patch is shown in Figure 2, along with a linear  -59-  Figure 1. Unitary NMDA currents in hippocampal neurons. (A) Typical unitary currents from an outside-out patch, taken from a hippocampal neuron exposed to 10 uM NMDA and 1 uM glycine (control solution) at the indicated V . (B) A record from the same p  patch after exposure to control solution with the addition of 20 uM Mg . 2+  Channel  openings are represented as downward deflections from a non-conducting baseline level.  - 60-  A -40 mV  4Ui  1 1 ui  ^ . . . u . i.»  ..L  -1.  -60 mV  3pA  B  Figure 1  10 ms  -61least-squares fit to the data points. The slope conductance (G) was 40 pS. Overall (n = 18 patches) the channel conductance was 44.4±4.1 pS at room temperature. Previous studies of outside-out patches from mouse central neurons have revealed the primary conductance state G for the NMDA channel of 40 - 50 pS (Ascher et al., 1988), as have investigations of cultured rat cortical neurons (Bertolino et al., 1988), and of cultured rat hippocampal neurons (Lester et ai, 1990). present experiments employed a higher external C a  2 +  It should be noted that the concentration (1.8 mM) than  used for the earlier estimates (1 mM); it has been shown that G is diminished by increased external C a  2 +  (Mayer and Westbrook, 1987). The reversal potential (V ), rev  i.e., the voltage at which there was no observed current, was extrapolated to +4.3±0.7 mV for n = 18 patches. This value for reversal potential is close to that documented in previous studies (Ascher et ai, 1988; Gibb and Colquhoun, 1991) and is consistent with the channel being non-selective for N a and K* (or Cs ). Further identification of +  +  the channel was done by adding 20 pM Mg * to the perfusion (bath) solution (Figure 2  1B).  The addition of M g  2+  resulted in rapid transitions between the open state and a  non-conducting, or blocked, state. The action of M g  2+  to cause a fast channel block of  the NMDA channel at hyperpolarizing potentials has been previously described (Mayer et ai,  1984; Nowak et ai,  1984).  As well, addition of 100 pM 2-amino-5-  phosphonovalerate (APV) to the external solutions eliminated the unitary currents (data not shown).  Previous work has shown that A P V acts as a competitive NMDA  antagonist (Davies et ai, 1981; Evans et ai, 1982). Occasionally, events of lower amplitude, i.e. with a conductance smaller than the primary conductance state of the NMDA channel, were observed in some patches, in both  control  conditions  and  in  the  presence  of  added  agents.  these  -  62-  Figure 2. Current-voltage relation for the patch shown in Figure 1 in control solution. The slope of the linear regression fit to the data points is the channel conductance (G), which was 40 pS for this patch; the intercept of the fitted line with the abscissa is the zero-current, or reversal, potential (V ), which was 4 mV for this patch. rev  - 63-  Figure 2  - 64openings (generally about 50% of the size of the larger openings, with a voltageindependent mean open time near 2.5 ms) likely represented a subconductance state of the NMDA ion channel. Although the low conductance events were not common, the use of single-channel recording allowed for isolation of the large conductance events and rejection of the relatively infrequent small conductance events. In the latter case, the macroscopic currents are a sum from all states of the system and would include contributions from states other than the primary conductance level. It should be noted that previous studies have also documented lower conductance NMDA substates in hippocampal (Jahr and Stevens, 1987) and cerebellar (Cull-Candy and Usowicz, 1987) neurons. In the present study, only events belonging to the primary large conductance state were included for analysis. The results obtained in control solution were then used as a basis for investigation of the properties of the potential neuroprotectant compounds L-687,384, DM, and (+)- and (-)-p-cyclazocine. The procedure was to apply 10 uM NMDA, along with 1 uM glycine, to the external face of outside-out patches, and, after recording at various patch potentials, to add one of the test compounds to the NMDA/glycinecontaining external solution and again to record at various patch potentials. As noted above, reduction of the frequency of events by compounds other than L-687,384 often prevented the recording of data over a wide range of voltages. In some cases, more than one concentration of the agents was applied.  The test compound was then  washed out, and recovery recordings, in the presence of NMDA and glycine, were made. Data obtained in the presence of drug were compared with control and recovery  - 65data in order to determine drug effects on the unitary properties of the NMDA ion channel. 3.2.1.2.Effects of the compounds on unitary NMDA current amplitudes and channel conductance  Typical unitary currents recorded from an outside-out patch in (A) control solution, (B) 1 pM (-)-p-cyclazocine and (C) 1 pM (+)-p-cyclazocine are shown in Figure 3.  The V was -40 mV and between (B) and (C), recovery data (wash-off of (-)-pp  cyclazocine) was obtained (not shown).  The amplitudes of the unitary currents in  control (A) and in the presence of the applied agents (B) and (C), as well as in the recovery between (B) and (C), were near 2.0 pA. From the traces in Figure 3 (A-C) it is evident that neither enantiomer of p-cyclazocine altered the heights of the unitary currents induced by NMDA. In order to quantitate the actions of (-)- and (+)-p-cyclazocine on the unitary conductance of the NMDA channel, distributions for channel amplitudes were constructed for currents recorded in the absence and presence of drug.  Typical  amplitude distributions are shown for control (Figure 4A), in the presence of 1 pM (-)-pcyclazocine (Figure 4B), and after exposure to 1 pM (+)-p-cyclazocine (Figure AC). As in Figure 3, current heights recorded with either drug present in the bath solution (up to 4 pM for (-)-p-cyclazocine and up to 10 pM for (+)-p-cyclazocine) were unchanged from control values over the voltage range studied (-100 mV < V < -20 mV). Hence, neither p  enantiomer of p-cyclazocine altered the amplitudes of unitary currents relative to control values.  -66-  Figure 3. Unitary NMDA currents before and after exposure to (-)- or (+)-|3cyclazocine in hippocampal neurons. (A) Typical unitary currents from an outside-out patch, taken from a hippocampal neuron, in control solution. (B) Openings from the same patch exposed to control solution with the addition of 1 uM (-)-p-cyclazocine. (C) Openings with the control solution containing 1 uM (+)-p-cyclazocine. contained 10 uM NMDA and 1 uM glycine; V  p  = -40 mV.  All solutions  Channel openings are  represented as downward deflections from a non-conducting baseline level.  - 67-  control  1 nM (-)-p-cyclazocine  B  1 n M (+)-(3-cyclazocine •  5 pA 10  ms  Figure 3  - 68Figure 4.  Amplitude distributions in the absence and presence of (-)- or (+)-P-  cyclazocine in hippocampal neurons. Unitary current amplitude distributions (V = -40 p  mV) are shown for the same patch as in Figure 4: (A) in control solution, (B) with 1 pM (-)-p-cyclazocine, and (C) with 1 pM (+)-p-cyclazocine. All solutions contained 10 pM NMDA and 1 pM glycine. Amplitude distributions were fit with Gaussian functions with means (A) 1.88 pA, (B) 1.86 pA, and (C) 1.92 pA. The number of events included in each histogram was 587 in (A), 112 in (B), and 253 in (C). The relatively small number of events in (B) is due to the effect of (-)-p-cyclazocine to lower opening frequency.  -69 "  160  Amplitude (pA) 140  Amplitude (pA)  Figure 4  - 70Figure 5. Current-voltage relation for the patch shown in Figures 3 and 4 exposed to 1 pM (-)-p-cyclazocine. The slope of the linear regression fit to the data points is G, 39 pS for this patch. The intercept of the fitted line with the abscissa is Vrev, 6 mV for this patch.  -  -80  7 1 -  -60  I  -40.  -20  0  /  I  V (mV) • -1 -2  • -3  I (PA)  Figure 5  - 72Figure 5 shows an IA/ plot for currents recorded from the patch shown in Figure 3 in the presence of 1 pM (-)-p-cycIazocine. For (-)-p-cyclazocine, as well as for cyclazocine (data not shown), the slopes of the l/V relations, as determined by linear least-squares fits to the points, corresponded to 40 pS conductances.  The  conductance (G) values for all patches were G = 40.6±2.5 pS for (-)-p-cyclazocine (n = 4) and G = 40.2±4.4 pS for (+)-p-cyclazocine (n = 5). A comparison with control data showed that neither enantiomer altered the conductance of the NMDA ion channel (see Section 3.2.1.1, Figure 2).  The extrapolated reversal potential (V ) for the patch rev  shown in Figure 5 was 4.0 mV, while for all patches V  rev  was 5.6±0.4 mV for  (-)-p-  cyclazocine (n = 4) and 4.0±0.5 mV for (+)-p-cyclazocine (n = 5), respectively, and unchanged from the value found in control (4.3 mV). Thus, channel selectivity, which in these experiments embraced inward N a and C a +  2 +  and outward Cs* flows, was not  altered by either enantiomer of p-cyclazocine. The data for current amplitudes, G, and V  rev  in control and in the presence of (-)- or (+)-p-cyclazocine are shown in Table 1. Table 1 summarizes the values for unitary NMDA properties in outside-out  patches from hippocampal neurons, for control solution and in the presence of all of the compounds studied. The amplitude measurements are the mean values of amplitude distribution means, while G and V  rev  are from linear least-squares fits to IA/ plots  constructed for all the patches exposed to a given drug. The number of patches tested with a given drug is given by n; means are ±S.E.M.  - 73Table 1.  Unitary NMDA current amplitudes (INMDA), conductances (G), and  reversal potentials (V ) for control solution and in the presence of the compounds rev  indicated, as measured in outside-out patches from hippocampal neurons.  ......G(P.SL.. V (mV)  compound control  V =-20 mV  V =-40 mV  V =-60 mV  V =-80 mV  Vp=-100mV  -1.12±0.09  -2.02±0.05  -2.74±0.07  -3.64±0.11  -4.75±0.22  44.4±4.1  +4.3±0.7  (-)-p-cyc  -0.93±0.17  -1.96±0.06  -2.84±0.27  -3.45±0.10  ND  42.2±2.5  +4.4±0.4  (+)-p-cyc  -1.08±0.01  -1.98±0.04  -2.67±0.17  ND  ND  43.3±4.4  +5.2±0.5  DM  ND  -2.07±0.04  -2.80±0.09  -3.64±0.20  ND  39.3±2.2  +12.3±0.7  L-687,384  ND  -1.93±0.11  -2.71 ±0.24  -3.56±0.34  -4.26+.0.91  39.2±8.1  +9.5±2.1  p  p  p  p  rev  All solutions contained 10 uM NMDA and 1 uM glycine, (-)-p-cyc = 1 uM (-)-pcyclazocine, (+)-p-cyc = 1 uM (+)-p-cyclazocine, DM = 5 uM dextromethorphan, and L687,384 = 20 uM L-687,384. Values are reported as ±S.E.M. ND = not determined.  - 74-  3.2.2. Summary of drug effects on unitary NMDA current amplitudes and channel conductance in hippocampal neurons None of the agents altered the conductance properties of the NMDA ion channel measured in Mg -free solutions. In control solution, the IA/ relation for the NMDA ion 2+  channel in the presence of 1.8 mM external C a  2 +  and in the absence of M g  2+  was linear  in the range -100 < V < -20 mV (Figure 2). This result is consistent with previous p  measurements of the conductance of NMDA single-channel behaviour in mouse cortical and cerebellar neurons (Ascher et al., 1988; Ascher and Nowak, 1988) and in rat hippocampal and cerebellar neurons (Jahr and Stevens, 1987; Gibb and Colquhoun, 1991). In the presence of (-)-p-cyclazocine (Figure 5), or in the presence of the other compounds, the IA/ relation was also found to be linear, showing no voltage-dependent action of the compounds on unitary NMDA conductance at the potentials studied. It should be noted that the potentials used in the study lie near the physiological resting potential and encompass potentials encountered during normal synaptic transmission; as well, these voltages are likely representative depolarized potentials that would be commonly encountered during  of the  pathological  situations where neuronal homeostasis is compromised (Parsons et al., 1993, 1995; Danysz et al., 1995).  Other aspects of unitary NMDA conductance were also  unchanged by the compounds. The ionic selectivity filter for NMDA ion channels is non-selective for Na* and Cs* (or K*) (Mayer and Westbrook, 1987b; Ascher et al., 1988; Ascher and Nowak, 1988). The NMDA ion channel is unusual among ligandgated ion channels in that it also has high permeability to C a  2 +  (Mayer and Westbrook,  1985; Ascher and Nowak, 1986; MacDermott et al., 1986), with the magnitude of the  -75C a * permeability relative to the permeability for Na* (PC/PHB) having a value of 10.4 2  (Mayer and Westbrook, 1987b).  However, since [Na*] was over 100 x [Ca *] at the 2  external face of the patch in these experiments ([Na*] = 140 mM, [Ca *] =1.8 mM), the 2  e  0  conductance properties of the NMDA ion channel were largely determined by the inward flow of Na*. In the present experiments, the concentrations of Na* and Cs* were identical on either side of the patch ([Na*] = 140 mM = 8  [Cs*]i)  and thus the zero-current  potential found from IA/ plots was close to 0 mV (Figure 3). The values obtained for G and Vrev were unchanged by any of the compounds, showing that these agents did not alter the properties of the channel elements responsible for determining ionic selectivity or channel conductance.  3.2.3. Drug actions on the kinetics of unitary NMDA currents in hippocampal neurons The principal aim of the electrophysiological studies was to characterize the kinetic interactions of the putative uncompetitive NMDA antagonists p-cyclazocine, dextromethorphan (DM) and L-687,384 with NMDA ion channels. The study of the kinetic behaviour of unitary currents requires the analysis of open and closed time distributions following the collation of a large number of events. This analysis would yield information on the effects of agents on mean open and closed times, frequency of channel opening, and channel open probability ( P ) . 0  3.2.3. LMean open time of single NMDA ion channels in control solution The mean open time (to ) is the arithmetic mean of the time each channel event P  dwelt at the open state level in the data records. The mean open time for the NMDA  -76ion channel in hippocampal neurons was measured from analysis of recordings from outside-out patches, such as the patch shown in Figure 1, and was found to be 6.2±1.0 ms at V = -40 mV (n = 17). Previous studies of NMDA ion channel kinetic behaviour p  have shown the mean open time to be near 6 ms at V = -60 mV in outside-out patches p  from mouse central neurons (Ascher and Nowak, 1987) and from rat cortical neurons (Bertolino et al., 1988). 3.2.3.2.Effects of the compounds on mean open time  As shown in Figure 3, the effects of (-)-p-cyclazocine on channel kinetics included a decrease in the mean open time (Up) of the ion channel. This effect could be reversed upon wash-out of the compound (not shown).  (+)-p-cyclazocine had little  effect on Up, although a small decrease in mean open time was observed at the highest concentration employed (10 uM). It can be noted that channel events in the presence of (-)-p-cyclazocine did not evince any rapid flickerings from open levels to a nonconducting state such as was found with M g  2+  block of the NMDA channel (see  Figure 1B). Typical channel open time distributions from the same patch as shown in Figure 3 are shown in Figure 6 for NMDA-induced channel openings in the absence of drug (A), in the presence of 1 uM (-)-p-cyclazocine (B), and in the presence of 1 uM (+)-pcyclazocine (C) (V = -40 mV). A single peak in each log open time distribution allowed p  each distribution to be fit with a single-exponential function. As exemplified in the patch in Figure 3, the mean open time with 1 uM (-)-p-cyclazocine was reduced relative to control. The mean of the control distribution in Figure 6A was 4.6 ms, while the mean of the (-)-p-cyclazocine distribution in Figure 6B was 3.2 ms.  At this same  - 77Figure 6.  Open time distributions in the absence and presence of (-)- or (+)-3-  cyclazocine in hippocampal neurons. Open dwell-time histograms are shown for the same patch as in Figures 3 - 5 (V = -40 mV) (A) in control solution, (B) with 1 pM (-)-fJp  cyclazocine, and (C) with 1 pM (+)-|3-cyclazocine; all bath solutions contained 10 pM NMDA and 1 pM glycine.  Open time distributions were fit with single-exponential  functions. The time constants of the fitted curves were 4.62 ms in (A), 3.23 ms in (B), and 4.37 ms in (C), with the same number of events being included in each histogram as in Figure 4.  - 78-  A  Log open time (ms)  B  Log open time (ms)  C  Log open time (ms)  Figure 6  - 79concentration, the (+)-enantiomer caused only a small decrease in mean channel open time in this patch; the mean of the (+)-p-cyclazocine distribution in Figure 6C was 4.4 ms. Overall, at V = -40 mV, mean open times were reduced to 74±15% of control by p  200 nM (-)-p-cyclazocine (n = 2), 65±6% of control by 1 pM (-)-p-cyclazocine (n = 4; significant at the P < 0.05 level), and 50±8% of control by 4 pM (-)-fJ-cyclazocine (n = 2). The (+)-enantiomer did not reduce mean open time at concentrations of 1 pM (n = 5; P = 0.17) or 4 pM (n = 3; P > 0.20); applying 10 pM (+)-p-cyclazocine produced a mean open time that was 81 ±17% of control (n = 2) at V = -40 mV. p  The effects of DM and L-687,384 also included reductions in NMDA ion channel mean open times in outside-out patches from hippocampal neurons. For the former compound, the mean open time was reduced to 84±4% of control with 5 pM DM (n = 4), 70±6% of control with 20 pM DM (n = 4), and 68±18% with 50 pM DM (n = 2), all at V = p  -40 mV. The reduction produced by 20 pM DM was significant (P = 0.02), while that produced by 5 pM DM was not (P = 0.17).  For the latter compound, at the same  potential, mean open times were reduced to 96±9% of control by 5 pM L-687,384 (n = 4) and 71 ±6% of control by 20 pM L-687,384 (n = 4); a single patch with 40 pM L687,384 exhibited a mean open time that was 64% of control. The reduction produced by 20 pM L-687,384 was significant (P = 0.02) while that produced by 5 pM L-687,384 was not (P > 0.20).  The effects of both agents on mean open time could be fully  reversed by washing out of the agents.  Fast transitions to the baseline (non-  conducting) level as evidenced with block of the NMDA ion channel by Mg * (see 2  Figure 1) were not observed for DM or L-687,384.  - 80Figure 7. Opening frequencies of NMDA ion channels in the presence of (-)- or (+)-p-cyclazocine in hippocampal neurons. The frequency of opening events relative to control, ±S.E.M., is shown for (-)-p-cyclazocine (solid bars; n = 2, 4, and 2 for 0.2, 1, and 4 uM (-)-p-cyclazocine, respectively) and (+)-p-cyclazocine (striped bars; n = 5, 3, and 2 for 1, 4, and 10 uM (+)-p-cyclazocine, respectively) in outside-out patches from hippocampal neurons; V = -40 mV. All solutions contained 10 uM NMDA and 1 uM p  glycine. * significant difference (P < 0.01) between control and drug.  -  81-  [ 8 - c y c l a z o c i n e ] (uM)  Figure 7  - 823.2.3.3. NMDA ion channel opening frequency in control solution  The effects of agents on the frequency of openings was determined. The procedure was to divide the total number of events by this recording period to obtain a value for mean frequency.  In n = 17 patches, the mean channel opening frequency  was 13.8±2.4 s" at V = -40 mV. 1  p  3.2.3.4. Effects of the compounds on channel openingfrequency  The (-)-enantiomer, and to a lesser extent, the (+)-enantiomer, of p-cyclazocine diminished the frequency of NMDA ion channel opening in a concentration-dependent manner (e.g. see Figure 3). This action was reversed by washing out the agents (not shown). The effects of p-cyclazocine on opening frequency are summarized in Figure 7 at V = -40 mV for both enantiomers at concentrations up to 4 uM. The decrease in p  opening frequency by these agents paralleled their action to decrease mean open time, although the effect of decreasing frequency was more pronounced than reduction of open time at any concentration tested. DM,  like  (-)-p-cyclazOcine, concentration-dependently  diminished  frequency, an effect that could be reversed by washing out.  opening  Channel opening  frequencies, at V = -40 mV, were 81 ±18% (n = 4), 65±46% (n = 4), and 32±10% (n = 2) p  of control in the presence of 5, 20, and 50 uM DM, respectively. The effect of DM on frequency was significant at 20 uM (P < 0.02) but not at 5 uM (P > 0.20). In contrast to (-)- and (+)-p-cyclazocine concentrations  and DM, L-687,384 had no effect on frequency at  up to 40 uM.  At 5 and 20 uM L-687,384  (V  p  = -40  - 83Figure 8. Open state probabilities for NMDA ion channels in the presence of (-)- or (+)P-cyclazocine in hippocampal neurons. Log concentration-response curves, where the response is the open state probability (P ), at V = -40 mV, normalized to the open state 0  p  probability in control solution, in outside-out patches taken from hippocampal neurons and in the presence of (A) (-)-p-cyclazocine and (B) (+)-p-cyclazocine.  The n values  for each concentration were the same as those given for mean open time (Section 3.2.3.2) and frequency (Section 3.2.3.4). The lines are least-squares logistic fits to the data. The ICso's, where P = 0.5, were 84 nM for (-)-p-cyclazocine and 14 pM for (+)-P0  cyclazocine. *P < 0.02.  - 84-  Figure 8  - 85mV), the frequency was 114±69% of control (n = 4) and 109±7% of control (n = 4); neither were significantly changed from control (P > 0.20 in both cases). 3.2.3.5. NMDA ion channel open state probability in control solution  The channel open state probability (P ), calculated from the product of mean 0  open time and mean opening frequency is an index of the kinetic behaviour of a channel. The mean control value for P in n = 17 patches was 0.082±0.013 for V = -40 0  p  mV, indicating the channel was in a conducting state approximately 8% of the time. At other V , open channel probabilities were calculated as 0.103±0.078 for V = -20 mV (n p  p  = 2), 0.094±0.025 for V = -60 mV (n = 14), 0.087±0.016 for V = -80 mV (n = 9), and p  p  0.105±0.010 for V = -100 mV (n = 2). P was not found to vary with V (P > 0.20 by p  0  p  one-way analysis of variance). 3.2.3.6. Effects of the compounds on open state probability  Figure 8 shows semi-log plots of open channel probability calculated at V = -40 p  mV for increasing concentrations of (-)-p-cyclazocine (A) and (+)-p-cyclazocine (B). P  0  values shown were calculated as the product of mean open time and channel opening frequency. To illustrate the actions of the agents on open state probability, the P value 0  for each patch has been normalized to its open state probability in control solution. Logistic fits to the data for each enantiomer are shown superimposed on the points. The plots in Figure 8 were then used to extrapolate ICso values for p-cyclazocine effects on P and the results showed an ICso of 84 nM for (-)-p-cyclazocine (V = -40 0  mV) and an  IC50  p  of 14 pM for (+)-p-cyclazocine (V = -40 mV). p  - 86The relationship between macroscopic and unitary (microscopic) currents is IMACRO = IUNIT  N  P , where IMACRO is the macroscopic whole-cell current, IUNIT is the 0  unitary current size, N is the number of conducting ion channels in the cell membrane, and P is the mean channel open state probability. An effect of a given agent on one of 0  the parameters on the right side of the equation would produce a proportional effect on IMACRO- Since neither (-)- nor (+)-|3-cyclazocine were found to alter the size of unitary NMDA currents (see Section 3.2.1.2), then, assuming no change in the number of conducting channels, the results for P yield 0  NMDA currents.  IC50  values for inhibition of macroscopic  The values also show the (-)-enantiomer to be about 170X more  potent than the (+)-enantiomer against NMDA ion channel open probability in hippocampal neurons at V = -40 mV. p  The effects of DM or L-687,384 were to concentration-dependently reduce P . 0  As with (-)-p-cyclazocine, DM's effect on P was more potent than its effect on either Up 0  or frequency. This was expected, since P is the product of Up and frequency, which 0  were both lowered by this agent.  On the other hand, the action of L-687,384 was  relatively weak and similar in potency to its effect on Up! this was due to its lack of effect on opening frequency. The P„ values for DM and L-687,384 were used to determine IC50  values for inhibition of NMDA channel open probability by these compounds.  These values were 4.4 uM for DM (V = -60 mV) and 61 uM for L-687,384 (V = -60 p  mV).  p  -87-  3.2.4. Summary of drug effects on unitary NMDA kinetics in hippocampa neurons DM, (-)- and (+)-p-cyclazocine, and L-687,384 reduced the mean open time and mean opening frequency of the NMDA ion channel in a concentration-dependent and reversible manner. The rank order of potency for this effect was (-)-p-cyclazocine > DM > (+)-p-cyclazocine > L-687,384. The open state probability was also reduced with the same rank order of potency; this was due to the effects of the agents on mean open time and an additional action, with the exception of L-687,384, of decreasing channel opening frequency.  L-687,384 did not reduce the rate of channel opening and  therefore had a relatively weak effect on P . For each agent, the reduction of P„ was 0  found to increase as V became more negative. The voltage-dependent actions of the p  four agents on single NMDA ion channel P suggest that these compounds would 0  exhibit voltage-dependent inhibition of NMDA activity at the whole-cell level, similar to what has been found for P C P (Honey et al., 1985; Bertolino et al., 1988), ketamine (Honey et al., 1985; MacDonald et al., 1987), MK-801 (MacDonald et al., 1991), and memantine (Chen et al., 1992; Parsons et al., 1993). The present results with (-)- and (+)-p-cyclazocine are the first to demonstrate voltage-dependence of benzomorphan actions against NMDA activity; similarly, this is the first demonstration of DM's voltagedependent blocking action, although voltage-dependence of the DM metabolite, dextrorphan, has been previously observed (Parsons etal., 1993).  3.2.5. Consideration of mechanism of uncompetitive antagonist action The overall results obtained with the 4 agents on single NMDA ion channels are now considered in order to arrive at a mechanism of action for inhibition of currents  - 88through NMDA ion channels. The rationale for this approach is that determination of the on- and off-rates of drug interactions with NMDA ion channels would enable predictions concerning their utility as putative neuroprotectants in vivo.  3.2.5. l.Lack of drug effects on unitary NMDA ion channel conductance  The results presented above are consistent with open channel block by the agents (-)- and (+)-p-cyclazocine, DM, and L-687,384. For example, the size of the unitary currents activated by NMDA and the conductance of the ion channel were unchanged by any of the agents, suggesting the channel was open prior to block. No fast transitions between open and non-conducting levels were observed; hence, the compounds did not exhibit a Mg -like open channel blockade. The reduced frequency 2+  of openings, as evident with all agents but L-687,384, would indicate relatively long interaction times with the NMDA receptor-ion channel complex.  The results also  showed the agents did not act to block and unblock the ion channel with extremely fast kinetics (i.e. numerous open-blocked transitions occurring within the 0.3 ms resolution time), since such rapid kinetics would appear as diminished heights of the unitary currents. In this regard, it should be noted that no apparent increase in baseline noise was evident upon application of any of the 4 agents; observation of increased baseline noise between otherwise typical channel openings could represent opening events that were blocked so fast as to have their full amplitude poorly resolved. Also, the agents had no effect on the ionic selectivity of the channel, since there was no difference in the reversal potential in the presence or absence of any of the compounds.  - 893.2.5.2.Decrease of mean open time and mean opening frequency by the compounds  The most salient indicator of open channel blockade was the concentrationdependent reductions in mean open time found with all four compounds. The decrease in the mean open time of the NMDA-activated unitary events by the compounds is in contrast to agents that exert inhibitory effects on receptor-coupled ion channels strictly through competition with the agonist at the receptor site (competitive antagonists); with the latter type of antagonist, no decrease in mean open time is associated with attenuation of channel activity (Katz and Miledi, 1971; Ascher et ai, 1988). In a model in which the agent exhibits a bimolecular interaction with its site and thus produces ion channel blockade, the reduction in frequency corresponds to the presence in the single-channel records of non-conducting periods when the agent occupies the site and blocks current flow.  Longer blocking periods would produce  greater reductions in frequency; hence, the potency of a given agent as a reducer of channel opening frequency is related to the length of time the agent occupies the blocker site. This feature of the compounds' interaction with NMDA ion channels is an important determinant of their actions to reduce P . It should be noted that it is possible 0  that the channel may close with the agent trapped inside, as has been demonstrated for MK-801 (Huettner and Bean, 1987) and memantine (Blanpied et ai, 1997). The extent to which this occurs with the present agents was not tested.  3.2.5:3.Applicability of open channel blockade model  An open channel blockade scheme can be represented by the following diagram  - 90-  where C is the ion channel in its closed state, 0 is the channel in the open state, and B is the channel in the blocked state. The rate constants are defined as ki = the rate constant for isomerization of the ion channel from the open to closed state, Id = the rate constant for isomerization of the ion channel from the closed to open state (the reverse of k^, k = the on-rate constant for the blocking agent, and k. = the off-rate 2  2  constant for the blocking agent.  Since the lifetime of a given state is equal to the  inverse of the sum of the rate constants leading away from that state, the following equation is applicable: (W)'  1  = k-1  +  MD].  Here (to )" is the inverse of the mean open time in the presence of a given 1  P  concentration ([D]) of the agent. Thus, a plot of (to )" vs. [D] would be linear with slope 1  P  k and ordinate intercept k.1 (Neher and Steinbach, 1978; Ascher and Nowak, 1988). 2  This analysis was applied to the 4 agents studied and the results are given below. 3.2.6. Analysis of data using an open channel blockade scheme 3.2.6.1.Determination of on-rate constants A plot of the inverse of mean open time vs. drug concentration at V = -40 mV is p  presented in Figure 9 for (-)-p-cyclazocine up to 4 uM (A) and for (+)-J3-cyclazocine up to 10 uM (B). Both plots were well-fit with linear relationships over the concentrations studied, indicating the applicability of the open channel blockade model with the two enantiomers of p-cyclazocine. The values for the onward (blocking) rate constant k were found to be 7 6 x 10 M* s" (n = 4) for (-)-p-cyclazocine, 6.0 x 10 M"V for (+)-p8  1  1  7  2  -91Figure 9. Determination of onward rate constants for (-)- and (+)-3-cyclazocine in hippocampal neurons.  Plots of 1/to vs. concentration are shown for (A) (-)-PP  cyclazocine (n = 4) and (B) (+)-|3-cyclazocine (n = 5) in hippocampal neurons; V = -40 p  mV. The lines shown are fits using linear regression analyses. Values for k are given 2  in the text.  - 92-  Figure 9  - 93-  Figure 10. Determination of onward rate constants for DM in hippocampal neurons. A plot of 1/top vs. concentration is shown for DM in a hippocampal neuron at the indicated V . The lines are fits using linear regression analyses. The values for k are given in p  the text.  2  - 94-  Figure 10  - 95Figure 11.  Determination of onward rate constants for L-687,384 in hippocampal  neurons. A plot of 1/to vs. concentration is shown for L-687,384 in a hippocampal P  neuron at the indicated V . The lines are fits using linear regression analyses. The p  values for k are given the text. 2  - 96-  600  r  "20  40  L 687,384 l M l H  Figure 1 1  - 97cyclazocine (n = 5). The mean open time in the absence of the drug could be found from extrapolation of the plots to the ordinate and inverting the intercept.  This  procedure yielded mean open times near 5 ms (V = -40 mV), close to those found from p  analysis of open times in the current records for control (Section 3.2.3.1). A similar open channel blockade analysis was applied to the actions of DM and L-687,384. The results are shown in Figure 10 for DM and Figure 11 for L-687,384; the plots were constructed using data from n = 6 patches for each compound. At V = -40 p  mV, the respective blocking rate constants k were 5.0 x 10 M" s' and 2.1 x 10 M" s' . 6  1  1  7  1  1  2  Thus, at a patch hyperpolarization of -40 mV the rank order for k values was (-)-p2  cyclazocine > (+)-P-cyclazocine > L-687,384 > DM. As evident in Figures 10 and 11, the results include V other than -40 mV. With DM, the plots also include V = -60 mV p  p  and -80 mV, and with L-687,384, plots are shown for -100 mV < V„ < -40 mV. Such plots were not possible with (-)-p-cyclazocine since the marked effect of this agent to reduce frequency did not allow a sufficient number of openings to be recorded at the different V . With DM, the k values were 8.5 x 10 M V at V = -60 mV, and 1.0 x 10 6  P  2  7  p  M ' V at V = -80 mV. A similar trend was found for L-687,384, where the kz values 1  p  were 3.6 x 10 M " V at V = -60 mV, 5.9 x 10 M ' V at V = -60 mV, and 8.9 x 10 M * V 7  7  7  p  p  a t V = -100mV. p  3.2.6.2.Determination of off-rate constants A determination of the magnitudes of k. (the off- or unblocking rate constant) is 2  not as straightforward as the quantitation of k . 2  In principle, two procedures can be  used to evaluate k . One method is to attribute fast channel flickering in single channel 2  1  - 98Figure 12. Determination off-rate constant k. in hippocampal neurons. Closed dwell2  time histograms are shown for a single outside-out patch isolated from a hippocampal neuron (V = -40 mV) (A) in control solution and (B) in the presence of 1 uM (-)-pp  cyclazocine; all bath solutions contained 10 uM NMDA and 1 uM glycine. Closed time distributions required 3 exponential terms for a satisfactory curve-fit. In (A) the closed time components were 0.41 ms, 4.0 ms, and 60.8 ms, while in (B), closed time components were 0.63 ms, 18.2 ms, and 388 ms, with the same number of events being included in each histogram as in Figures 4 and 6.  - 99-  Figure 12  -100records to drug actions and determine mean values of times during the flickering episodes. Since none of the compounds evinced this type of behaviour, an analysis of this sort was not possible. A second procedure is to compare closed time distributions between controls and drug and relate any differences in time components to the unblocking effect of the agent. This second approach was used in this work. One complication in using closed-time distributions for the analysis of k. is that 2  in controls such distributions exhibited several time components. In these experiments, closed time distributions were best fit with 3 components. Previous studies have also reported multiple closed time component distributions in hippocampal neurons (Gibb and Colquhoun, 1992; Jahr and Stevens, 1990; Gibb, 1989) and in cerebellar granule cells (Howe etal., 1988, 1991). A comparison of closed time distributions in control solution and in the presence of (-)-p-cyclazocine or DM indicated that the primary effect of the compounds was to produce a prolonged third time component. This result was used to estimate values for k.2 for the two compounds. Figure 12 shows the closed time distributions for a typical patch under control conditions (A) and following the addition of 1 pM (-)-p-cyclazocine. The distribution in Figure 12A was fit using 3 components with values 0.41 ms, 4.0 ms, and 60.8 ms. Overall, in the control, the smallest time component was in the range 0.4 -1.0 ms, with two other components, an intermediate component in the 2 - 1 0 ms range and a slow component in the 50 - 80 ms range, also being evident. The effects of (-)-pcyclazocine on closed time distributions are indicated in Figure 12B (V = -40 mV); p  here, the 3 closed time components were 0.63 ms, 18.2 ms, and 388 ms. The slow time component in closed time distributions for (-)-p-cyclazocine was in the 200 - 400  -101ms range.  For simple open-channel blockade, this time component represents the  introduction of non-conducting states, by the agent, of mean length 200 - 400 ms and served to obscure the slow time component observed in control closed time distributions.  The enhancement of this later component was reflected in the lower  event frequency observed in the presence of (-)-p-cyclazocine, and is presumably a reflection of the time the agent associated with its blocking site. The value of this time component is the inverse of the off-rate, or unblocking, constant for the agent. Thus, k. was 2.5 - 5.0 s" for (-)-p-cyclazocine at V = -40 mV (n = 4). 1  2  p  The dissociation  constant Ko is calculated as the ratio k. /k . For (-)-p-cyclazocine, this quantity was 3 2  2  7 nM (n = 4). While this value is lower than the  ICso  for (-)-p-cyclazocine reduction of  P (84 nM), it is worthwhile noting that the former calculation is subject to greater error 0  than the latter, since the k term itself has limitations in reliability due to the complexity 2  of closed time distributions. It should be noted that an effect of (-)-p-cyclazocine addition in some patches, such as that shown in Figure 12, resulted in a prolongation of the intermediate time component. This change was not as consistent as the effect of the agent on the late component, nor was the change generally as large as that for the third time component. The view taken with regard to this observation was that the introduction of a large component by addition of the agent tended to artefactually increase the value of the intermediate time component determined by the fit. However, the possibility cannot be excluded that (-)-p-cyclazocine may have an effect on the closed time distributions in addition to the action of introducing a long time component as described above.  -102The effect of DM of altering closed time distributions was smaller than for (-)-pcyclazocine, i.e., the change in the long time component was not as great with the former agent compared to the latter. A slow time component in the range 80 - 1 0 0 ms was evident in closed time distributions for DM, which allows k. for DM to be estimated 2  at 10 - 1 3 s" (V = -60 mV; n = 6). Using the rate constants k_ and k , a Ko of 1.2 - 1.5 1  p  2  2  uM was estimated for DM interaction with NMDA ion channels (n = 6). Conversely, distributions for L-687,384 were not changed from control, a reflection of this compound's lack of effect on opening frequency. It was assumed that the k. for L2  687,384 was sufficiently high that the association time for this agent with the NMDA ion channel was of similar value to one of the closed time components observed in control distributions.  This last point was reflected in the lower potency of this agent as a  reducer of NMDA ion channel P . 0  -103-  CORTICAL NEURONS 3.2.7. Drug actions on unitary NMDA current amplitude and channel conductance in cortical neurons Cortical neurons were also studied in the present work since the differential distribution of NMDA receptor subunits in the C N S , as well as variations in tissue susceptibility to excitotoxic damage, suggests that there may exist differences in NMDA receptor-ion channel properties between tissues (Baumgarten and Zimmerman, 1992; McBain and Mayer, 1984; Danysz et al., 1995).  For example, the stoichiometry of  NMDA receptor subunit expression exhibits some degree of variation in both recombinant and wild type receptors (McBain and Mayer, 1994), producing varying sensitivity to both agonist and antagonist actions (McBain and Mayer, 1994).  NMDA  receptors from different areas of the C N S may have varying subunit compositions (McBain and Mayer, 1994); thus, the properties of active NMDA receptors as well as their antagonist sensitivity may depend on their location within the C N S . Since L687,384 and the (+)-enantiomer of 3-cyclazocine were found to exhibit low potency as NMDA antagonists in hippocampal neurons, only (-)-p-cyclazocine and DM were studied in cortical neurons.  In this section, the results with (-)-p-cyclazocine are  presented for illustrative purposes while the results with DM are summarized in tabular form. The organization of this section follows that outlined above in the analysis of hippocampal neurons.  -1043.2.7.1. Amplitudes of unitary NMDA currents and channel conductance in control solution Typical unitary currents recorded from rat cortical outside-out patches exposed to NMDA and glycine are presented in Figure 13 (V = -60 mV) in the presence of 10 p  pM NMDA and 1 pM glycine. An IA/ curve was constructed from this patch and is shown in Figure 14. The slope conductance was 44 pS and the zero-current potential was +12 mV, values similar to those obtained in the hippocampal neurons and to those obtained in previous studies of NMDA receptor-ion channel properties (Ascher et al., 1988; Bertolino et al., 1988; Gibb and Colquhoun, 1991). The values for G and V  rev  (extapolated) for all patches in control solution were 44.2±3.9 pS and +11.9±1.1 mV (n = 7). 3.2.7.2. Effects of (-)-B-cyclazocine and DM on unitary NMDA amplitudes and channel conductance Typical unitary currents recorded in the presence of 200 nM (-)-p-cyclazocine are shown in Figure 15; the data are from the same patch as shown in Figure 14. The amplitude distributions from this patch are shown for control (Figure 16A) and for 200 nM (-)-p-cyclazocine (Figure 16B), both at V = -80 mV. Here, the distribution means p  were 4.6 pA in control and 4.7 pA in the presence of (-)-p-cyclazocine. The IA/ plot for this patch in the presence of 200 nM (-)-p-cyclazocine is shown in Figure 17. (-)-pcyclazocine had no effect to alter the conductance of the NMDA channel (conductance in the presence of 1 pM (-)-p-cyclazocine = 40 pS or the ionic selectivity (zero-current potential in the presence of 200 nM (-)-p-cyclazocine = +14 mV). The values for G and  -105Figure 13.  Unitary NMDA currents in cortical neurons.  Shown are typical  unitary currents from an outside-out patch, taken from a cortical neuron, exposed to 10 uM NMDA and 1 uM glycine (control solution) at the indicated V . Channel openings p  are represented as downward deflections from a non-conducting baseline level.  -106-  -40 mV  -60 mV  -80 mV  3pA 10 ms  Figure 13  -107Figure 14.  Current-voltage relation for the patch shown in Figure 13 in control  solution. The slope of the linear regression fit to the data points is G, which was 44 pS for this patch; the intercept of the fitted line with the abscissa is for this patch.  V v, ra  which was 12 mV  -108-  Figure 14  -109Figure 15. Unitary NMDA currents after exposure to (-)-p-cyclazocine in cortical neurons. Shown are openings from the same patch as in Figure 13 exposed to control solution with the addition of 200 nM (-)-p-cyclazocine at the indicated V . Channel p  openings are represented as downward deflections from a non-conducting baseline level.  ' -110-  -40  mV  V  -60 mV  i -80 mV  3pA 10 ms  Figure 15  -111Figure 16. Amplitude distributions in the absence and presence of (-)-p-cyclazocine in cortical neurons. Unitary current amplitude distributions (V = -80 mV) are shown for p  the same patch as in Figures 1 3 - 1 5 : (A) in control solution, and (B) during exposure to 200 nM (-)-p-cyclazocine. All solutions contained 10 uM NMDA and 1 uM glycine. Amplitude distributions were fit with Gaussian functions with means (A) 4.59 pA and (B) 4.71 pA. The number of events included in each histogram was 362 in (A) and 267 in (B).  -112-  Figuro 16  -113Figure 17. Current-voltage relation for the patch shown in Figures 1 3 - 1 6 during exposure to 200 nM (-)-p-cyclazocine. The slope of the linear regression fit to the data points is G, which was 40 pS for this patch; the intercept of the fitted line with the abscissa is V , which was 14 mV for this patch. rev  -114-  Figure 17  -115V  rev  for all patches were 39.5±6.1 pS and +14.7±1.9 mV (n = 4), respectively. The  amplitudes at various V , conductance, and zero-current potential is shown for control p  solution and in the presence of either (-)-p-cyclazocine or DM in Table 2 for cortical neurons.  No significant difference in the amplitudes at a given potential was found  between control and either of the two compounds (P > 0.05 by two-way analysis of variance.  3.2.8. Summary of drug effects on unitary NMDA amplitudes and channel conductance in cortical neurons IA/ relations for unitary currents activated by NMDA in cortical neurons in 1.8 mM external C a  2 +  and in the absence of M g  2+  were found to be linear, consistent with  previous descriptions of NMDA single-channel behaviour in mouse cortical and cerebellar neurons (Ascher et al., 1988; Ascher and Nowak, 1988) and in rat hippocampal and cerebellar neurons (Jahr and Stevens, 1987; Gibb and Colquhoun, 1991).  Neither (-)-p-cyclazocine, at concentrations up to 4 pM, nor DM, at  concentrations up to 20 pM, altered the conducting properties of the NMDA ion channel in the range -80 mV < V < -40 mV, where l/V relations maintained linearity in the p  presence of either compound. The mean amplitude of the unitary currents measured at a given V were the same in the presence of either agent as in the control; values for p  slope conductance and V (Figure 14, Table 2) were also unchanged from the control. p  These results showed that, as for hippocampal neurons, DM and (-)-p-cyclazocine had no effect on the channel elements responsible for ionic selectivity and conductance.  -116Table 2. Unitary NMDA current amplitudes (INMDA), conductances (G), and reversal potentials (V ) for control solution and in the presence of the compounds indicated, as rev  measured in outside-out patches from cortical neurons.  V„=-20mV  V =-40 mV  v =-60mV  V =-80mV  G (pS)  control  1.39±0.14  -2.36±0.09  -3.27±0.17  -3.97±0.35  44.2±3.9  +11.9±1.1  (-)-p-cyc  - 1.52±0.02  -2.27±0.21  -3.35±0.41  -4.47±0.33  49.1 ±9.5  +9.0±1.7  DM  ND  -2.28±0.24  -3.10±0.40  -3.97±0.64  42.3±7.7  +13.8±2.5  p  p  p  V  r o  (mV)  All solutions contained 10 uM NMDA and 1 uM glycine. (-)-f3-cyc = 200 nM (-)-{}cyclazocine, and DM = 5 uM dextromethorphan. The data for control are the means of 7 patches, while data for (-)-p-cyclazocine and DM are each the means of 3 patches. Values are reported as ±S.E.M.  -117-  3.2.9. Drug effects on channel kinetics in cortical neurons 3.2.9.1.Effects of (-)-j$-cyclazocine and DM on unitary NMDA mean open time The mean open time for n = 7 patches from cortical neurons was 6.1 ±0.4 ms at V = -60 mV. Figure 15 shows that (-)-p-cyclazocine decreased the open-state dwell p  time of NMDA-activated unitary currents. This effect was concentration-dependent and reversible (not shown).  Fast transitions (flickering) between the open level and a  nonconducting state, as found with M g  2+  block of the NMDA channel (Mayer et al.,  1984; Nowak et ai, 1984), were not observed with (-)-P-cyclazocine. Figure 18 shows typical channel open time distributions (from the same patch as shown in Figures 13 and 15) for channel openings in the absence (A) and in the presence of 200 nM (-)-pcyclazocine (B) for V  p  = -80 mV.  Open time distributions were fit with single-  exponential functions. The mean of the control distribution in Figure 18A was 2.2 ms, while the mean of the (-)-p-cyclazocine distribution in Figure 18B was 1.8 ms. Overall, mean open times were reduced to 78±1% with 200 nM (-)-p-cyclazocine (n = 3; significant with P = 0.04) and to 59±18% of control with 1 uM (-)-p-cyclazocine (n = 2) a t V = -80mV. p  DM was also found to reduce the mean open dwell times of NMDA-activated unitary currents in cortical neurons. Mean open time was 68±18% of control at V = -80 p  mV (n = 3; significant at P = 0.05); at the same V , a single patch exposed to 20 uM DM p  had an observed mean open time that was 35% of control. No flickering between open and closed states, such as found with fast block by Mg *, was observed. 2  -118Figure 18. Open time distributions in the absence and presence of (-)-p-cyclazocine in cortical neurons.  Open dwell-time histograms are shown for the same patch as in  Figures 13 - 1 7 (V = -80 mV): (A) in the control and (B) with 200 nM (-)-p-cyclazocine. p  All bath solutions contained 10 pM NMDA and 1 pM glycine. Open time distributions were fit with single-exponential functions. The time constants of the fitted curves were 2.17 ms in (A) and 1.78 ms in (B), with the same number of events being included in each histogram as in Figure 16.  -119-  A  Open time (ms)  Figure 18  -1203.2.9.2.Effects of (-)-B-cyclazocine and  DM on unitary NMDA opening  frequency  The effects of (-)-p-cyclazocine on the mean frequency of channel opening was studied for cortical neurons. The procedures were the same as outlined in Section 3.2.3.4 for hippocampal neurons where the total number of events was divided by total recording time (at least 10 s) to obtain a mean frequency. In n = 7 patches exposed to control solution, the mean frequency of channel opening was 9.2±3.0 s" at V = -60 1  p  mV. (-)-P-cyclazocine  reversibly  and in a  concentration-dependent  manner  diminished the mean frequency of opening in cortical neurons, although this effect was not found to be significant (P = 0.08 at 200 nM (-)-p-cyclazocine). The reduction of the mean frequency of unitary current events by 200 nM (-)-p-cyclazocine is illustrated in Figures 13 and 15. Figure 19 shows the effect of (-)-p-cyclazocine to reduce frequency over the concentration range 0.05 - 4 pM (V = -60 mV). p  The reduction of channel opening frequency by DM was also observed in cortical neurons. In n = 3 patches (V = -60 mV) exposed to 5 pM DM, the frequency p  was 74±23% (significant at P = 0.05) of that observed in control solution; in a single patch exposed to 20 pM DM, the opening frequency was 57% of that observed in the same patch exposed to control solution.  -121Figure 19.  Opening frequencies of NMDA ion channels in the presence of (-)-p-  cyclazocine in cortical neurons.  The frequency of opening events, normalized to  control, is shown for 0.05, 0.2, 1, and 4 uM (-)-p-cyclazocine (n = 1, 3, 2, and 1, respectively) in outside-out patches from cortical neurons; V = -60 mV. All solutions p  contained 10 uM NMDA and 1 uM glycine. *P = 0.08.  -122-  Figure 19  -1233.2.9.3.Effects of (-)-B-cyclazocine and DM on unitary NMDA channel open probability  The mean values of P , the product of mean opening frequency and mean open 0  time, for NMDA-activated ion channels in cortical neurons were 0.035±0.023 (n = 2), 0.054±0.010 (n = 7), 0.033±0.017 (n = 6), and 0.064±0.029 (n = 3) at V = -20 mV, -40 p  mV, -60 mV, and -80 mV, repectively. P was not dependent on V in cortical neurons. 0  p  The P values determined in cortical neurons were lower than the corresponding values 0  determined for hippocampal neurons (see Section 3.2.2.7), although this difference was significant only for V = -40 mV and V = -60 mV. p  p  The actions of (-)-p-cyclazocine and DM on P were assessed by determination 0  of changes in the product of mean open time and mean opening frequency relative to control. Figure 20 shows a semi-log plot of open channel probability (P ) at V = -40 0  p  mV for increasing concentrations of (-)-p-cyclazocine, with a logistic fit to the data shown superimposed on the points. The P values have been normalized to the control 0  open state probability calculated for each patch. The plot in Figure 20 was used to determine an  IC50,  which in this case was 680 nM for (-)-p-cyclazocine inhibition of P in 0  outside-out patches from cortical neurons. Assuming that (-)-p-cyclazocine did not alter the effective number of conducting channels in the cell membrane, and using the result that amplitudes of unitary currents were not changed by the agent, then the single channel results predict that a (-)-p-cyclazocine concentration of 680 nM would attenuate macroscopic NMDA responses by a factor of 50% at a potential of -40 mV. Reduction in P by DM was also calculated, and it was found that, in n = 3 0  patches, 5 uM DM produced a P that was 47±17% of that in control solution (V = -60 0  p  -124Figure 20. Open state probability of NMDA ion channels in the presence of (-)-pcyclazocine in cortical neurons.  Log concentration-response curves, where the  response is the open state probability (P ), at V = -60 mV, normalized to the open state 0  p  probability in control, in outside-out patches taken from cortical neurons in the presence of (-)-p-cyclazocine. The n values for each concentration are the same as those given for mean open time (Section 3.2.9.1) and frequency (Section 3.2.9.2). The lines are least-squares logistic fits to the data. The ICso, where P = 0.5, was 305 nM 0  for (-)-p-cyclazocine. *P < 0.05.  -125-  1.50  r  o D_  0.01  0.1  1  [(-)-B-cyclazocine] (pM)  Figure 20  10  -126mV); the single patch tested with 20 pM DM exhibited a P that was 41% of that 0  observed in control solution for that patch (V = -60 mV). As only two concentrations of p  DM were tested on cortical neurons, it was not possible to construct a full concentration-response curve for P  0  vs. [DM].  However, using the results quoted  above, an ICso for DM inhibition of NMDA P„ of 3.8 pM (V = -60 mV) was estimated. p  3.2.10.Summary of drug effects on kinetics of unitary NMDA currents in cortical neurons The actions of (-)-p-cyclazocine and DM on unitary NMDA current kinetics were to reduce both the open time and the frequency of opening of the ion channel. These changes resulted in a concentration-dependent decrease in channel open state probability, which would predict a similar reduction in macroscopic currents mediated through NMDA receptor-ion channels, (-)-p-cyclazocine was found to be ~10X more potent than DM in reducing P . The actions of the two agents on channel kinetics in 0  cortical neurons were similar to those observed in hippocampal neurons.  3.2.11 .Applicability of open channel block model in cortical neurons The results of the study on cortical neurons can be interpreted in the same manner as described previously for the agents' action on hippocampal neurons: that is, open channel blockade underlies the effects of the compounds on NMDA-activated unitary currents. The lowering of P by the agents was due to decreases in both top and 0  frequency. The decrease in top is characteristic of uncompetitive antagonism via open channel blockade, as competitive antagonism is not associated with alterations in mean open time (Katz and Miledi, 1972; Ascher and Nowak, 1988). As noted in Section 3.2.5.3, the relationship between concentration and mean channel open time for an  -127open-channel blocker is described by the equation (Up)' = k-i + k [D] (Neher and 1  2  Steinbach, 1978). An analysis for the rate constants k and k. is presented below and 2  2  follows the procedures outlined above (see Section 3.2.6).  3.2.12.Analysis of cortical neuron data using an open-channel block scheme 3.2. J 2. J.Determination of on-rate constants  Plots of (Up)' vs. [D] are shown in Figure 21 with (-)-p-cyclazocine and in Figure 1  22 with DM. The values for k found from the slopes of these plots were 12.4 x 10 M' 6  2  1  s ' for (-)-p-cyclazocine (Figure 21; V = -40 mV) and 7.9 x 10 M" s' for DM (Figure 1  6  1  1  p  22; V = -60 mV). The patch with (-)-p-cyclazocine was sufficiently stable to obtain data p  at several V and produced k values of 7.7 x 10 M ' V for V = -20 mV, 13.5 x 10 M ' V 6  p  1  2  6  1  p  for V = -60 mV, and 31.2 x 10 M ' V for V = -80 mV (not shown). The increase in k 6  p  p  2  with patch hyperpolarization suggests the site of action of these agents to be located within the electrical field of the membrane, such as within the channel pore.  An  interesting result is that despite a large difference in potency between (-)-p-cyclazocine and DM (in excess of tenfold) there was little difference between the onward (blocking) rate constants k . 2  This finding would suggest that the potency for open channel  blockade is not mediated by differences in the blocking rate constant.  3.2.12.2.Determination of off-rate constants  Closed time distributions were analyzed in order to estimate the unblocking rate constant k_ for (-)-p-cyclazocine. While the effect of (-)-p-cyclazocine to reduce 2  -128Figure 21. Determination of the onward rate constant for (-)-p-cyclazocine in cortical neurons.  A plot of 1/to vs. concentration is shown for (-)-fJ-cyclazocine (n = 4) in  cortical neurons; V  P  p  = -40 mV.  The lines shown are fits using linear regression  analyses. Values for k are given in the text. 2  -129-  soo r  400 h  100 h  0  I  1  1  1  1  1  0  1  2  3  4  5  [(-)-R-cyclazocine] (uM)  Figure 21  -130Figure 22. Determination of the onward rate constant for DM in cortical neurons. A plot of 1/top vs. concentration is shown for DM (n = 5) in cortical neurons; V = -60 mV. p  The line is a fit using linear regression analyses. Values for k are given in the text. 2  -131-  500 r  200 X  100 h  0  5  10  15  [DM] (uM)  Figure 22  20  25  -132Figure 23.  Determination of off-rate constant k. in cortical neurons. 2  Closed time  histograms are shown for a single outside-out patch isolated from a cortical neuron (V  p  = -80 mV) (A) in control solution and (B) with 200 nM (-)-p-cyclazocine; all bath solutions contained 10 uM NMDA and 1 uM glycine. Closed time distributions required 3 exponential terms for a satisfactory curve-fit.  In (A), the closed time components  were 0.96 ms, 7.9 ms, and 147 ms, while in (B), closed time components were 0.45 ms, 15.9 ms, and 448 ms, with the same number of events being included in each histogram as in Figures 16 and 18.  -133-  A 80-  -  1  0  1  2  Log closed time (ms)  B 50r  Log closed time (ms)  Figure 23  3  *  -134-  frequency in most cases made the analysis of closed time distributions in the presence of the agent impossible, one patch was sufficiently stable to derive distributions with enough events to provide a reliable analysis. Figure 2 3 shows typical closed time histograms from the same outside-out patch, taken from a cortical neuron, as is shown in Figures 1 3 - 1 8 in control solution (A), and in the presence of 2 0 0 nM (-)-Pcyclazocine (B). Closed time distributions in both the presence and the absence of drug were fit with three-component exponential equations.  A longer slow time  component was evident in distributions for (-)-p-cyclazocine when compared to control, while the other two time components were unaffected.  The value of this time  component were near 4 0 0 ms, which presumably represents the mean time the agent interrupted ion flow through the channel; this corresponds to a k. of 2 . 5 s' at V = - 8 0 1  2  p  mV. The dissociation constant KD, calculated as the ratio k-2/k , .was 8 0 uM for (-)-p2  cyclazocine at this V . p  3.2.13.Conclusions regarding open-channel block analysis in both neuronal types Several observations concerning the actions of the tested agents on NMDAactivated unitary currents have been applied to generate a model for the antagonism of NMDA receptors by the compounds: (i) the lack of effect of either agent on NMDA channel conductance, (ii) concentration-dependent inhibition of NMDA open state probability by the agents, with the action of reducing the mean dwell time spent in the open state and of introducing non-conducting level "gaps" in the current records (and thus decreasing frequency) being the primary determinants of the agents' effect of reducing P , (iii) the presence of a prolonged exponential component in closed time 0  -135distributions for the applied agents that was not present in control, leading to a decreased frequency of opening, and (iv) the linear relationship between (top)" and 1  concentration of the applied agent. All of these results are consistent with and support the conclusion that the compounds (-)-p-cyclazocine and DM inhibit current through NMDA ion channels via an open channel block mechanism. The basis for drug actions is not likely due to differences in k since, especially for cortical neurons, onward rate 2  constants were similar in magnitude. Instead, the  IC50  values for inhibition of P were 0  close to the Ko values found by dividing k. by k and it is suggested that k. is a major 2  2  2  determinant of the potency for a given agent. 3.2.14. Biophysical probes: (i) studies with Mg * in hippocampal neurons 2  3.2.14.1.Interaction of DM with the Mg * site 2  A single experiment using M g  2+  was used to yield information as to the channel  site for binding of DM. As noted previously, the divalent cation Mg * interacts in a 2  voltage-dependent manner with the NMDA ion channel (Nowak et ai, 1984; Mayer et ai, 1984). Experiments in which 20 pM M g  2+  was added to the bath solution containing  NMDA and glycine resulted in fast transitions between the open level and a blocked state such that the channel mean open time was reduced by 60% at V = -80 mV p  (Figure 1B). This pattern of activity is consistent with previous observations of Mg * 2  actions on NMDA ion channels (Nowak et al. 1984; Ascher and Nowak, 1988). In an outside-out patch from a hippocampal neuron, channel kinetics were studied in control conditions, in the presence of 20 pM added Mg *, in 20 pM DM, and, finally, in 20 pM 2  DM and 20 pM Mg . The concentrations of Mg * and DM chosen were those which, 2+  2  -136when applied separately, would decrease mean open times to approximately one-half of control magnitude at V = -60 mV; the concentration of the former agent was also p  well below those which produce other (enhancing) effects on NMDA ion channel activity, such as an increase in glycine affinity (Wang and MacDonald, 1995), a reduction of unitary current amplitude (Wang and MacDonald, 1995), and an increase in open channel probability (Paoletti et al., 1995). When M g  2+  was added (Figure 1B),  the mean open time was reduced by 49% (V = -60 mV) and by 64% (V = -80 mV). p  p  With DM added to the control solution, the mean open time was reduced by 52% (V = p  60 mV) and by 50% (V = -80 mV). DM further reduced mean open time when added to p  the solution containing M g ; at V = -60 mV the decrease was 66% and at V = -80 mV 2+  p  the decrease was 72%.  p  In addition, the frequency of openings was considerably  reduced when DM was added to the control solution with Mg . If the decrease in mean 2+  open time produced by each agent were mediated through independent sites, one would expect the fractional inhibitions to be additive, i.e. values of 76% for V = -60 mV p  and 82% for V  p  = -80 mV in the presence of the two agents should have been  observed. However, the values obtained show the effects of the agents to be less than additive, suggesting that the inhibition of mean open time by both agents was mediated by the same site, or a common locus was involved in the action of both agents. However, the deviation from additivity was not great (< 10%), making a definite conclusion from the available data impossible.  Further experiments regarding this  matter are required in order to ascertain whether a common site is involved in the actions of DM and M g  2+  on NMDA ion channels.  -137-  3.2.15.Biophysical probes: (ii) temperature studies with outside-out patches from cortical neurons In order to further investigate the biophysical interaction of uncompetitive NMDA antagonists with their site of action, variable temperature studies were carried out on outside-out patches derived from cortical neurons.  The use of temperature as a  biophysical probe has not often been used and it was reasoned that thermodynamic analysis could be used to help elucidate some of the mechanisms involved in uncompetitive NMDA blockade. 3.2.15.1.Dependence of unitary NMDA current amplitudes on temperature in control solution  Preliminary experiments were carried out in order to investigate the effects of changing temperature on unitary NMDA currents. Decreasing the bath temperature of a cortical outside-out patch preparation from 24°C to 14°C diminished the amplitude of unitary currents activated by 10 pM NMDA (Figure 24). The use of 24°C was chosen as this was close to room temperature and thus the data acquired at this temperature is comparable to all the other data in this work; cooling by 10°C was chosen in order to minimize unresolvable fast transitions. The Qio values for temperature dependence of current size were found by taking the ratio of current amplitudes at 24°C to current amplitudes at 14°C. The Q  1 0  values were found to be 1.4±0.1 at V = -60 mV (n = 6); p  refer to Table 3 for Qio's at other V . Q i values of this size indicate that NMDA unitary p  0  current heights were not strongly dependent on temperature.  The value for  conductance (G) in cortical patches held at 24°C was 47±4 pS (n = 6), while G at 10°C  -138Figure 24. Dependence of unitary NMDA currents on temperature. Shown are unitary currents in an outside-out patch taken from a cortical neuron at 24°C (A) and 14°C (B) at V = -60 mV. The external bathing solution contained 10 pM NMDA and 1 pM p  glycine.  -139-  A  B  24°C  14°C  Figure 24  -140Table 3. Q10 values for channel amplitudes in the absence and presence of DM. V (mV)  -40 mV  -60 mV  -80 mV  control  1.42±0.05  1.41±0.06  1.73±0.01  DM  1.37±0.11  1.60±0.05  1.99  P  Q  1 0  values shown are the ratio of channel amplitude at 24°C to that at 14°C as  determined in outside-out patches from cortical neurons. The concentration of DM was 5 pM. The control data were from n = 6 patches; the DM data were from n = 3 patches, except the value at V = -80 mV, which was a single observation. Means are ±S.E.M. p  -141was 35±6 pS (n = 6), corresponding to a Q10 of 1.4. A Qio of 1.4 strongly suggests that rate of ion flux through the channel is determined chiefly by free diffusion and is not affected by non-diffusible, non-ionic elements such as protein gates. Previous work on measurements of conductance in frog node of Ranvier has shown this quantity to generally have low sensitivity to temperature, with Q 's in the range 1.3 - 1.5 (Hille, 10  1975). The present results are also similar to findings for calcium-activated potassium channels in inside-out patches from cardiac myocytes (McLarnon and Wang, 1991) and ATP-dependent potassium channels, also in inside-out patches from cardiac myocytes (McLarnon et al., 1993), where respective Q  1 0  values for conductance were 1.4 and 1.5.  3.2. J5.2.Dependence of NMDA  ion channel kinetics on temperature in  control solution  Cooling outside-out patches from 24°C to 14°C increased the time spent in the open state of unitary currents activated by 10 pM NMDA. The ratio of the mean open time at 14°C to that at 24°C was taken as the d o for NMDA ion channel mean open time.  The Qio for this effect was 2.7±0.3 (V = -60 mV; n = 6), indicating that the p  channel spent, on the average, 2.7 times longer in the open state at 14°C than at 24°C (Table 4). A clear conclusion from these Q  1 0  measurements for amplitude and mean  open time is that the behaviour of these two parameters is governed by different ratelimiting processes. The frequency of single-channel events activated by 10 pM NMDA was diminished upon cooling the outside-out patches from 24°C to 14°C (Figure 24). The mean event frequency of outside-out patches exposed to 10 pM NMDA was 8.7±2.8 s"  1  -142Table 4. Q10 values for channel mean open times in the absence and presence of DM. V (mV)  -40 mV  -60 mV  -80 mV  control  2.82+0.26  2.71±0.30  3.98±0.75  DM  2.33±0.34  2.89±0.64  2.77  P  Q  10  values shown are the ratio of channel mean open time at 14°C to that at  24°C as determined in outside-out patches from cortical neurons. The concentration of DM was 5 pM. The control data were from n = 6 patches; the DM data were from n = 3 patches, except the value at V = -80 rriV, which was a single observation. Means are p  ±S.E.M.  -143at V = -60 mV. Cooling from 24°C to 14°C reduced the event frequency to 3.5±1.9 s'  1  p  at the same V . p  3.2.15.3.Effects ofDMon temperature modulation of channel behaviour  In 3 cases, the viability of the outside-out patches was preserved for long times and allowed a measurement of the temperature dependence of the actions of DM on the same patches as studied in the controls. At 14°C, the amplitude of NMDA-activated unitary currents and channel conductance were not affected by DM, whereas mean open time and event frequency were diminished by DM. It was found that the  Qi 's 0  for  mean open time in the presence of DM were similar to the Q ' s obtained in control 10  solution (Table 4).  Therefore, it may be stated that the actions of DM on channel  conductance and mean open time were independent of temperature. However, these data must be interpreted with some degree of caution, as the low opening frequency at low temperature meant that distributions of amplitude and mean open time were less than adequate in terms of containing sufficient events for reliable data analysis. 3.2.16. Summary of the use of biophysical probes to elucidate drug actions The decrease in mean open time produced by DM or Mg * are each mediated by 2  separate sites. If the two agents were indeed acting at the same intrachannel site, the decrease in mean open time expected upon addition of one agent to control solution would have the same value as the decrease expected upon addition to a solution containing the other agent.  Exposing an outside-out patch to the agents together  produced an effect that was less than the addition of the effects of the two agents  -144alone. This infra-additivity is also suggestive of an inhibition of one of the agents' actions by the other, as if DM occludes access by M g  2+  to its site, or vice versa.  The low Q ' s for unitary current amplitudes and conductance found in the 10  present study indicate that NMDA ion channel conductance was not strongly dependent on temperature.  The values obtained are suggestive of a process rate-limited by  aqueous diffusion of ions and not by, for example, a process involving changes in protein conformation such as a gating protein.  On the other hand, the higher Qio's  obtained for mean open time provide evidence that the channel elements responsible for channel gating are indeed rate-limited by non-diffusible species, e.g. protein-based gating structures. While the results with DM are somewhat difficult to interpret, the fact that addition of this agent did not change the Q ' s for channel amplitude and mean 10  open time suggests that uncompetitive antagonist action did not involve an additional process involving channel proteins or other non-solubilized structures. The conclusion derived from these experiments is that the mechanism of NMDA channel blockers is rate-limited by diffusion of these agents to their site of action. 3.3.  Microspectrofluorimetric studies  Microspectrofluorimetric studies using hippocampal and cortical neurons loaded with the Ca *-sensitive dye fura-2 were carried out: (1) to assess drug effects on 2  NMDA-induced increases in [Ca *],, this last quantity being particularly important in 2  terms of neurotoxicity mediated through NMDA receptors (Choi, 1988; Meldrum and Garthwaite, 1990); and (2) to assess drug specificity in terms of actions on other glutamate receptor types, i.e. NMDA vs. kainate or A M P A receptors, and on other C a * 2  permeable ion channels, i.e. NMDA vs. voltage-activated C a * channels. 2  Following  -145identification of potential neuroprotectants using electrophysiological means, further information regarding their actions is necessary in order to elucidate how their application affects neuronal functioning. The technique of optical recording from cells loaded with the Ca -sensitive dye would thus provide a useful supplement to the 2+  electrophysiological experiments in this regard. Note that depolarization mediated via non-NMDA receptors, as with A M P A and kainate receptor activation, would also result in inward C a  2 +  flux through voltage-activated C a  2 +  channels; however, studies have  shown that some forms of the A M P A receptor can also conduct C a (McBain and Mayer, 1994).  2 +  into the cell  As in the electrophysiological work, two tissue types,  specifically hippocampal and cortical neurons, were employed, due to recent evidence that the subunit composition and thus properties of NMDA receptors may vary throughout the C N S (McBain and Mayer, 1994; Danysz etal., 1995).  3.3.1. Uncompetitive inhibition of NMDA responses in hippocampal neurons 3.3.1.1.B-cyclazocine blockade of NMDA-evoked [Ca Jj responses: use2+  dependence [Ca ]j responses to 20 pM NMDA were tested in the absence and presence of (2+  )-p-cyclazocine, from 0.2 - 5 pM, or (+)-p-cyclazocine, 5 pM. In these experiments, 20 pM NMDA evoked [Ca ]j rises in hippocampal neurons of 481 ±46 nM from a baseline 2+  of 74±2 nM (n = 452). The NMDA-induced rise in [Ca *]j began approximately 10 s 2  after injection of NMDA into the chamber and persisted for about 30 s following washout. The return to baseline [Ca ]j was complete about 3 min following washout; 2+  the time course of [Ca *]j was similar in all cultures. 2  (-)-P-cyclazocine reduced the  -146Figure 25.  Attenuation of NMDA-evoked rises in [Ca ]j by f3-cyclazocine in  hippocampal neurons.  2+  Transient increases in intracellular calcium were evoked by  injections of 20 pM NMDA (N). Superfusion of 5 pM (-)-p-cyclazocine (right-hand solid horizontal bar) use-dependently attenuated the calcium response to the NMDA injections while superfusion of 5 pM (+)-fj-cycla*zocine (left-hand screened horizontal bar) had relatively little effect.  The inhibition of the NMDA response by (-)-p-  cyclazocine in this experiment was 92.8% at the 2  nd  NMDA application and the steady-  state inhibition produced by (+)-J3-cyclazocine was 20.5% at the 4  th  NMDA application.  This record is a mean of data taken from 28 neurons simultaneously. The use of 5 pM as a concentration  allows comparison between the two enantiomers, as this  concentration with (-)-p-cyclazocine nearly abolishes the NMDA responses while with (+)-P-cyclazocine the NMDA response are almost unaffected at this concentration. Inset: (-)-p-cyclazocine concentration-dependently inhibited [Ca ]i rises evoked by 2+  NMDA (data ±S.E.M. from 3 culture batches at each concentration). The logistic fit to the data produced an ICso value for (-)-p-cyclazocine of 272 nM.  -147-  700 • s  I !  s  calcii  5 >iM (-)-6-cyclazoclne  5 pM (*)-0-cyclazoclne  600 500 1  lular f i  a 400 e 0.0  300 -  ean intrt  <o u  at t.a [(-)-a-cydazocine)  io.o  200 • 100 !•  N N N 0  1000  N N N N N N 2000  3000  N N N N 4000  5000  time (s)  Figure 2 5  N N 6000  N N N N N 7000  8000  N N 9000  10000  -148NMDA-evoked rises in [Ca ]j in a concentration-dependent manner (Figure 25, left and 2+  centre).  The action of the drug on NMDA responses was use-dependent, so that  several (usually 5 or 6) agonist applications were necessary in order to observe a steady-state drug block. Similarly, several NMDA applications after drug washout were also required before the response stabilized again (Figure 25, right). The development of drug blockade was not dependent on time: initial responses to NMDA following superfusion of hippocampal neuronal cultures with (-)-p-cyclazocine for periods of > 1 hr, without agonist applications, were the same as the initial response in cultures in which the superfusion had lasted only 5 minutes (not shown). The number of agonist applications in the presence of (-)-p-cyclazocine required to reach a steady-state response level was inversely dependent on the drug concentration; that is, the higher the concentration of (-)-p-cyclazocine, the fewer the applications of NMDA that were necessary to achieve steady-state blockade.  The mean number of applications  necessary to achieve equilibrium block is given in Table 5, along with the degree of reduction of NMDA responses measured at equilibrium blockade. An  IC50  value of 272  nM for (-)-p-cyclazocine blockade of NMDA-evoked [Ca *]j increases in hippocampal 2  neurons was calculated from these results. Due to the low potency of (+)-p-cyclazocine determined electrophysiologically in hippocampal neurons, only one concentration (5 uM) was tested.  Neither enantiomer of p-cyclazocine altered resting [Ca ]i in  hippocampal neurons.  2+  -149Table 5. Reduction of responses to NMDA by p-cyclazocine in hippocampal neurons.  [(-)-p-cyclazocine]  n  mean # to ss  % blockade  #  (MM) 0.2  110  6.3  37.3±4.5  6  0.5  70  6.0  66.2±8.4  6  1  80  4.3  86.3±0.5  4  5  115  2.0  92.9±0.1  2  5 pM (+)-pcyclazocine  41  1.0  15.9±4.7  1  The percentage difference in steady-state blockade between control and during superfusion with the indicated concentration of (-)-p-cyclazocine, as measured from the #th NMDA application, is given as % blockade. The results with 5 pM (+)-p-cyclazocine are given for comparison. The mean number of NMDA applications required to reach steady state blockade is given as mean # to ss. The total number of neurons tested with each drug concentration is given as n. Means are ±S.E.M. of 3 culture batches.  -1503.3. 1. 2.DM effects on NMDA-evoked [Ca *]i responses 2  The effect of DM on NMDA-evoked rises in [Ca ]i was also studied in 2+  hippocampal neurons. In all hippocampal neurons tested, DM (1 - 50 pM; n > 34 at each concentration)  reduced NMDA-evoked rises in [Ca ]j in a concentration2+  dependent manner, with 5, 20 and 50 pM DM reducing NMDA-evoked responses by 55±1% (n = 127), 88±1% (n = 74) and 97±1% (n = 145), respectively. An  ICso  value of  4.1 ±0.2 pM was calculated from these results. In contrast to (-)-fJ-cyclazocine, there was no use-dependence observed in DM blockade of NMDA responses: the second response to NMDA in the presence of a given concentration of DM was always the same size as the first. Therefore, the first NMDA response following commencement of DM superfusion was used as the measurement of drug action. DM had no effect to alter resting [Ca ]i in hippocampal neurons. 2+  3.3.1.3.L-687,384 effects on NMDA-evoked [Ca ] 2+  t  responses  The effect of L-687,384 on [Ca Jj responses to boli of NMDA was tested in 2+  cultures of hippocampal neurons loaded with the calcium-sensitive fluorophore fura-2. L-687,384 (1-100 pM) produced a concentration-dependent and reversible reduction in the rise in [Ca ]i evoked by NMDA with an apparent 2+  ICso  value of 49±8 pM (n > 40 at  each concentration tested), with no effect on resting [Ca ]j. 2+  At the highest  concentration tested (100 pM), L-687,384 produced a 67±1% reduction of NMDAevoked rises in [Ca ]i {n = 81). The potency of L-687,384 as an antagonist of NMDA2+  evoked rises in [Ca ]j is in good agreement with the electrophysiological data, in which 2+  -151Figure 26.  Selectivity of (-)-p-cyclazocine for NMDA responses in hippocampal  neurons. Hippocampal neurons were challenged with either 50 mM K* (K*), 20 uM NMDA (N), 80 uM kainate (K), or 40 uM AMPA (A). The horizontal bar indicates the presence of 5 uM (-)-p-cyclazocine in the external perfusion medium. NMDA-evoked [Ca ]i 2+  rises were significantly affected by the agent (P < 0.001; analysis of variance  followed by Tukey test) while high-K\ kainate, and AMPA responses were minimally affected. This record is a mean of data obtained from 17 neurons simultaneously.  -152-  5 |iM (-)-p-cyolazocln«  400  h  K* N K A  0  1000  K ' N N K A  2000  3000  K* N N  4000  time (s)  Figure 26  K  A N N  5000  6000  7000  -153channel open probability was reduced by approximately 50% by L-687,384 at 60 uM. Since this potency was low, no further experiments were performed with L-687,384.  3.3.2. Drug effects on calcium responses to raised hC in hippocampal neurons 3.3.2.1. Effects of (-J-B-cyclazocine on [Ca ]j reponses to raised IC 2+  Neuronal depolarization, such as that induced by raising [K*] , has been shown to raise e  [Ca ]i 2+  in neurons by allowing entry through voltage-activated, L-type calcium channels  (Fletcher et ai, 1993; Church et ai, 1994a).  Application of 50 mM K* in the p-  cyclazocine experiments produced a [Ca ]i rise of 612±31 nM in hippocampal neurons. 2+  A s with NMDA applications, the [Ca ]j response began approximately 10 s after 50 mM 2+  K* injection into the chamber and persisted for about 30 s following washout. However, the relaxation of [Ca ]i to baseline was faster following K* washout than following 2+  NMDA washout (Figure 26). Responses to raised K* in the presence of 5 uM (-)-pcyclazocine were 96.9±8.5% of control in hippocampal neurons.  Thus, (-)-p-  cyclazocine had no effect to inhibit L-type calcium channel activity in hippocampal neurons at a concentration that abolished responses to NMDA.  3.3.2.2. Effect of DM on [Ca ]j responses to raised K* 2+  Superfusion of 50 uM DM attenuated the increase in [Ca ]i produced by 2+  application of 50 mM K* by 40% in hippocampal neurons (Figure 27).  In other  experiments, where more than one application of 50 mM K* was tried following commencement of DM superfusion, it was found that steady-state antagonism was achieved at the first 50 mM K* application (not shown); therefore, the action of DM to  -154Figure 27. Selectivity of DM for NMDA responses in hippocampal neurons. The effect of DM applied for the period indicated by the bar above the trace, on  [Ca ]i 2+  rises  evoked by 50 mM K* (K*), 20 pM NMDA (N), 80 pM kainate (K), or 40 pM AMPA (A) is shown. The response to NMDA was almost abolished, whereas responses evoked by K \ kainate, and AMPA were each reduced by approximately 45%. This record is a mean of data obtained from 5 neurons simultaneously.  -155-  Figure 27  -156block voltage-gated C a  2 +  channels was not use-dependent.  The action of DM to  antagonize voltage-gated calcium channels was in contrast to (-)-p-cyclazocine, which was ineffective against [Ca ]i responses to raised K*. 2+  The action of DM to inhibit  responses mediated by voltage-activated calcium channels was not unexpected, as this action has been previously observed electrophysiologically in cultured cortical neurons (Netzer et al., 1993) and in DM antagonism of rC-stimulated ^ C a * uptake into brain 2  synaptosomes (Carpenter et al., 1988).  3.3.3. Drug effects on non-NMDA responses in hippocampal neurons 3.3.3. J.Effects of (-)-B-cyclazocine on [Ca *]! responses to non-NMDA 2  agonists The effects of (-)-p-cyclazocine on [Ca ]i responses to 80 pM kainate and 40 pM 2+  A M P A were examined in cultured neurons.  Both excitants activate separate non-  NMDA ion channel subtypes and produce much of their [Ca ]j response by 2+  depolarizing the neurons and allowing C a  2 +  flux through voltage-activated  Ca  2+  channels, although there is evidence that some forms of A M P A receptor-ion channels are permeable to C a  2 +  (Danysz et ai, 1995). Application of 80 pM kainate produced  [Ca ]i rises of 270±67 nM in hippocampal neurons, whereas 40 pM A M P A produced 2+  [Ca ]j rises of 253±11 nM in hippocampal neurons. 2+  Figure 26 shows that, as with  raised K \ 5 pM (-)-p-cyclazocine had no effect to attenuate [Ca *]j responses to non2  NMDA agonists; responses to kainate and A M P A were 114.1±12.8% and 99.1±9.6%, respectively, of control in hippocampal neurons. These results show (-)-p-cyclazocine to be selective for the NMDA subtype of glutamate receptor in hippocampal neurons.  -1573.3.3.2.EJfects of DM on fCa J responses to non-NMDA agonists 2+  i  The actions of DM to affect [Ca ] rises evoked by the non-NMDA excitants 2+  f  A M P A and kainate were tested in hippocampal neurons. Reductions in 80 uM AMPAand 40 uM kainate-evoked responses by DM were, respectively, 4±2% and 0±3% (5 uM), 16±2% and 16±4% (20 uM), and 41 ±2% and 44±3% (50 uM) in hippocampal neurons (Figure 27). The reductions of AMPA- and kainate-evoked responses by DM paralleled the reduction of rises in  [Ca ]j 2+  evoked by 50 mM K* and thus reflect  blockade by DM of high voltage-activated C a  2 +  channels rather than non-NMDA  receptor antagonist activity (see Netzer et al., 1993; Church et al., 1994), although contributions from calcium-permeable states of the AMPA receptor (Danysz et al., 1995) cannot be definitely ruled out.  It is therefore likely that, at the concentrations  employed in the single channel studies (5, 20 and 50 uM), the action of DM was predominantly selective for the NMDA subtype of glutamate receptor.  3.3.4. Drug blockade of NMDA ion channels in cortical neurons 3.3.4. l.B-cyclazocine blockade of NMDA-evoked [Ca ]j responses 2+  Cortical neurons were also used to study the time course of the development of NMDA antagonism by (-)-p-cyclazocine.  [Ca ]i 2+  responses to NMDA in cortical neurons  were similar to those observed in hippocampal neurons. In cortical neurons, NMDA at 20 uM induced mean rises in 531). This rise in  [Ca ]j 2+  [Ca ]j 2+  of 360±60 nM from a baseline of 75±6 nM (n =  began seconds after injection of NMDA into the chamber and  persisted for about 30 s following washout.  Return to baseline  [Ca *]j 2  was generally  complete approximately 3 min following washout, (-)-p-cyclazocine effects on NMDA  -158Figure 28. Reduction of 20 uM NMDA-evoked [Ca ]j responses by p-cyclazocine in 2+  cortical neurons. Values shown are the steady-state means of measurements taken from three different cortical culture batches for each concentration tested, corrected for baseline [Ca ]j (see Section 2.3.1.3) and normalized to concentration responses with 2+  20 uM NMDA only; error bars refer to the S.E.M. for the normalized values of 3 culture batches. The total number of neurons tested under each treatment (grouping together the three culture batches) is indicated in parentheses above each bar.  -159-  Figure 28  -160-  Table 6. Reduction of NMDA responses by p-cyclazocine in cortical neurons n  mean # to ss  % blockade  #  0.02  43  1.0  13.4±1.4  4  0.1  56  1.0  28.6±5.9  4  0.2  68  6.4  50.2±0.7  6  0.5  86  5.8  73.9±5.3  6  1  107  4.4  83.3±6.4  4  5  65  2.0  92.212.1  2  5 M M (+)-B-  46  1.0  21.5±2.4  1  [(-)-P-cyclazocine] (MM)  cyclazocine The percentage difference in steady-state blockade between control and during superfusion with the indicated concentration of (-)-p-cyclazocine, as measured from the #th NMDA application, is given as % blockade. The results with 5 pM (+)-p-cyclazocine are given for comparison. The mean number of NMDA applications required to reach steady state blockade is given as mean # to ss. The total number of neurons tested with each drug concentration is given as n. Means are ±S.E.M.  -161responses in cortical neurons were similar to those observed in hippocampal neurons, in terms of use-dependence and in level of blockade at equilibrium. Figure 28 shows the concentration-response curve for p-cyclazocine inhibition of NMDA responses in fura-2-loaded cortical neurons, while the values obtained at each concentration are shown in Table 6, along with information pertaining to the use-dependence of (-)-pcyclazocine.  Low concentrations  reached  equilibrium  immediately  following  commencement of (-)-p-cyclazocine superfusion; mean # to ss (steady state) is hence labelled 1.0. The ICso value for (-)-p-cyclazocine inhibition of NMDA-induced [Ca ]i 2+  increases in cortical neurons was 220 nM. 5 pM (+)-p-cyclazocine decreased NMDA responses by 21.5±2.4% in cortical neurons (not shown).  Neither enantiomer of p-  cyclazocine affected baseline [Ca ]i in cortical neurons. 2+  3.3.4.2.DM blockade of NMDA-evoked [Ca ]  responses  2+  t  As shown in Figure 29, DM acted to decrease [Ca ]j reponses to NMDA in 2+  cortical neurons.  No use-dependence was observed with DM blockade of NMDA-  evoked responses in cortical neurons.  NMDA responses were reduced to 72±6%,  46±7%, 19±5%, and 4.4±0.2% of control by 2, 5, 20, and 50 pM DM, respectively (n = 75). An ICso value of 5.4 pM was obtained for inhibition of NMDA-evoked [Ca ]i rises in 2+  fura-2-loaded cortical neurons, which is similar to the value obtained for DM inhibtion of NMDA-evoked rises in [Ca ]j in hippocampal neurons, 4 pM. DM had no effect to alter 2+  resting [Ca *]j in cortical neurons. 2  -162Figure 29.  Reduction of 20 uM NMDA-evoked [Ca ]j responses by DM in fura-22+  loaded cortical neurons. DM was applied for the period indicated by the bar above the trace at the concentrations shown. The n for each treatment is indicated inparentheses above each bar. simultaneously.  This record is a mean of data obtained from 22 neurons  -163-  1000 SO j/m DM  800  2  §  =  s  « 2 c — c  (0  E .2 .2 (0 «  600 400 200  o  0  N N N N N N N N  2000  NN  4000  time (s)  Figure 29  N N N  6000  8000  -1643.3.5. Drug effects on calcium responses to raised hC in cortical neurons 3.3.5.1. Effect of (-)-P-cyclazocine on [Ca ] reponses to raised K* 2+  t  Application of 50 mM K* evoked [Ca ]i rises of 302±18 nM in cortical neurons (n 2+  = 71). Figure 30 shows the effect of 5 pM (-)-3-cyclazocine on [Ca ]j responses to 50 2+  mM K* in cortical neurons. Here, the responses to 50 mM K* were not affected by this concentration of (-)-p-cyclazocine, whereas in contrast the NMDA-evoked response was eliminated by the second application of agonist. Responses to raised K* in the presence of 5 pM (-)-p-cyclazocine were 102.0±2.2% of control in cortical neurons, indicating that, as with hippocampal neurons, (-)-P-cyclazocine has no effect on voltage-sensitive C a  2+  channels in cortical neurons.  3.3.5.2. Effect of DM on [Ca J responses to raised K* 2+  t  [Ca ]i responses to raised K* (50 mM) in the absence and presence of DM are 2+  shown in Figure 31. In contrast to the lack of effect produced by (-)-p-cyclazocine against voltage-activated C a  2 +  channels, 50 pM DM inhibited K*-evoked rises in [Ca ]i 2+  to 63.7±6.5% of control in cortical neurons. DM inhibition of neuronal C a  2 +  channels  has been documented previously (Carpenter et al., 1988; Netzer et al. 1993). 3.3.6. Drug effects on non-NMDA responses in cortical neurons 3.3.6. J.Effects of (-)-B-cyclazocine on [C^Jt  responses to non-NMDA  agonists Figure 30 also shows kainate- and AMPA-evoked [Ca ]j rises in the absence 2+  and presence of (-)-p-cyclazocine.  Injection of 80 pM kainate into the chamber  -165Figure 30. Selectivity of 5 uM (-)-p-cyclazocine in cortical neurons. [Ca ]j responses 2+  to 50 mM potassium (K*), 20 uM NMDA (N), 80 uM kainate (K), and 40 uM A M P A (A) are shown for control, during superfusion of 5 pJVI (-)-p-cyclazocine (solid bar), and after washout of (-)-p-cyclazocine.  Responses in the presence of (-)-p-cyclazocine  were 86.8%, 30.8%, 8.2%, 109.6%, and 106.9% of control for K*. N, N, K, and A, respectively. simultaneously.  This trace is the mean of responses from 27 neurons measured  -166-  5 p M (->-0-cycluoclna  700  « fe §  600 500  =5 ~ 400 u E S-2 300  .£ -a  c o 200  <o  » E  100 0  K+NKA  K+NNKA  K+ N N K A N N  2000  4000 time (s)  Figure 30  6000  N N  8000  -167Figure 31.  Responses to raised K* and non-NMDA agonists in the absence and  presence of DM in cortical neurons.  [Ca ]j 2+  responses to 50 mM potassium (K*), 20 uM  NMDA (N), 80 uM kainate (K), and 40 uM A M P A (A) are shown for control, during superfusion of 50 nM DM (solid bar), and after washout of DM. Responses in the presence of DM were 57.2%, 15.9%, 103.2%, and 84.7% of control for K*. N, K, and A, respectively. simultaneously.  This trace is the mean of responses from 19 neurons measured  -168-  1000  2000  3000  time (s)  Figure 31  4000  5000  6000  -169produced [Ca ]j rises of 146±23 nM in cortical neurons. 40 pM A M P A produced [Ca ]i 2+  2+  rises of 83±7 nM in cortical neurons. Superfusion of (-)-p-cyclazocine, at 5 pM, had little effect to alter these rises. Responses to kainate and AMPA were 94.5±2.2% and 99.4±0.3% of control, respectively. These results are in agreement with those obtained in hippocampal neurons in that they show (-)-p-cyclazocine to be selective for the NMDA subtype of glutamate receptor. 3.3.6.2.Effects of DM on [Ca *]/ responses to non-NMDA agonists 2  Following the same procedure as with the hippocampal neurons, DM was tested for its actions on [Ca ]j responses to the non-NMDA agonists kainate and AMPA. Responses 2+  to 80 pM kainate or 40 pM AMPA were reduced to 96.0±7.3% and 89.2±4.5% by 20 pM DM in cortical neurons (Figure 31). The reductions in responses to kainate and AMPA were smaller than the reductions produced by DM against responses to 50 mM K*, suggesting that the [Ca ]i responses produced by kainate and AMPA in cortical 2+  neurons, unlike hippocampal neurons, were not mediated directly through DM-sensitive Ca  2 +  channels. In any case, DM's actions against glutamate receptors were restricted  to the NMDA subtype. 3.4.  Neuroprotection studies  3.4.1. (-)-ff-cyclazocine as a candidate neuroprotectant The action of (-)-p-cyclazocine to block the NMDA ion channel, as found in the electrophysiological and microspectrofluorimetric  experiments, would favour  possibility of its use as a clinical neuroprotective agent.  the  Selectivity for NMDA vs.  calcium channels was much more pronounced with (-)-p-cyclazocine than with DM, an  -170agent that has been previously shown to have neuroprotective properties in vitro (Choi, 1987) and is currently undergoing clinical trials against stroke (Parsons et al., 1995; Danysz et al., 1996).  Therefore, it was appropriate to test the ability of (-)-J3-  cyclazocine to protect neurons in vitro in order to support its identification as a candidate neuroprotectant. Cultured rat hippocampal and cortical neurons, prepared in the  same  manner  microspectrofluorimetric  as  those  studies,  employed (see  in  Methods)  the were  electrophysiological used  to  assess  and the  neuroprotective properties of (-)-p-cyclazocine against NMDA excitotoxicity. 3.4.2. Neuroprotection studies with hippocampal neurons 3.4.2. J.Excitotoxicity produced by NMDA in vitro  In the absence of drug, hippocampal cultures exposed to 1 mM NMDA + 1 mM glycine exhibited a marked decrease in the number of cells extruding trypan blue dye. Neurons that took up the dye following excitotoxic challenge displayed signs of severe neurotoxicity.  For example, the plasma membranes appeared rough and granular,  axonal processes were either discontinuous or not present at all, and vacuole formation could clearly be observed in the cytoplasm. As well, the overall morphology of the cells most affected by the treatment was severely distorted. These neuronal somata lost their characteristic bipolar or pyramidal shape, and instead were rounded and swollen to greater than twice the average volume observed prior to treatment. The survival rate of cultured hippocampal neurons following 24 hr exposure to 1 mM NMDA was 29.7±5.2% (Figure 32).  Addition of 1 uM glycine only, with no NMDA, produced  cultures that 24 hr later appeared healthy and morphologically similar to those  -171Figure 32. Protection against NMDA excitotoxicity by p-cyclazocine in hippocampal neurons. Cultured hippocampal neurons were exposed to culture medium containing 1 mM NMDA plus 1 uM glycine, with or without either (-)-p-cyclazocine (filled bars) or (+)P-cyclazocine (grey bars). Cell survival was assessed 24 hours later.  Bars indicate  percentage of surviving neurons present at 24 hours relative to the amount present immediately preceding NMDA exposure. The mean values (±S.E.M., n = 8) for cultures containing (-)-p-cyclazocine are represented by the solid bars, while the striped bars represent the means (±S.E.M., n = 8) obtained from cultures to which had been added (+)-P-cyclazocine.  *P < 0.05; **P < 0.001 as determined by one-way analysis of  variance followed by the Tukey test.  -172-  Figure 32  -173observed before the treatment.  Experiments where no NMDA was used showed a  survival rate of 72.7+4.4% for hippocampal cultures over the 24 hr period.  3.4.2.2. Neuroprotection by (-J-B-cycJazocine in hippocampal neurons  Addition  of (-)-p-cyclazocine  concentration-dependently  protected  cultured rat  hippocampal neurons from morphological damage induced by 1 mM NMDA (Figure 32). 67.3+2.1% (n = 8) of cells survived following NMDA + 1 pM (-)-p-cyclazocine challenge, compared to 29.7±5.2% surviving after challenge with NMDA alone.  10 pM (-)-p-  cyclazocine almost completely protected the culture from NMDA-induced excitotoxicity (73.8±4.3% cells surviving; n = 8). The majority of neurons observed following treatment with NMDA + 10 pM (-)-p-cyclazocine appeared healthy and morphologically similar to those observed before treatment; these cells did not take up the trypan blue stain.  No obvious vacuolization in the cells extruding the stain was observed,  indicating (-)-p-cyclazocine itself was not toxic to the neurons at the concentrations used (Olney et al., 1989).  The extrapolated ICso for (-)-p-cyclazocine inhibition of  NMDA-induced excitotoxicity in hippocampal neurons was approximately 0.9 pM. Raising the glycine concentration to 10 pM did not alter the effectiveness of (-)-pcyclazocine in hippocampal neurons (data not shown).  3.4.2.3. Effect of (+)-B-cyclazocine in hippocampal neurons  The neuroprotective properties of the (+)-enantiomer were also studied. While the (+) enantiomer was considerably less active than the (-)-enantiomer, some degree of protection was seen with 10 pM (+)-p-cyclazocine. In hippocampal neurons, survival rate with 10 pM (+)-p-cyclazocine was 48.5±6.2% while survival with 1 pM (+)-p-  -174Figure 33.  Stereospecific neuroprotection by p-cyclazocine in cortical neurons.  Shown is the percentage of neurons protected from 1 mM NMDA + 1 uM glycineinduced neuronal degeneration by either (-)-p-cyclazocine or  (+)-p-cyclazocine,  measured at 24 hours. Values were corrected for procedural cell losses (as estimated from control cultures where no NMDA or p-cyclazocine was added) and normalized to the mean values obtained from cultures where only 1 mM NMDA + 1 uM glycine was added. All treatments were performed in parallel in 7 different culture batches; each treatment was replicated 3 times within a given culture batch.  Error bars indicate  S.E.M. for the culture batches. Statistics were performed as a one-way Analysis of Variance followed by a Tukey test. *P < 0.05; **P < 0.005; ***P < 0.001.  -175-  Figure 3 3  -176cyclazocine was 37.5±5.1% (Figure 32).  Note that 1 uM (+)-J3-cyclazocine afforded  almost no protection against NMDA-induced neurotoxicity.  The low potency of (+)-p-  cyclazocine in the neuroprotection assay paralleled the potency difference between (-)and (+)-p-cyclazocine observed in the electrophysiological experiments. 3.4.3. Neuroprotection  studies with cortical  neurons  3.4. 3. 1.Effects of NMDA only Similar to hippocampal neurons, cortical neurons were also severely damaged by NMDA, showing a survival rate of 32.0±3.8% after 24 hr exposure to 1 mM NMDA (Figure 33). The majority of cells present 24 hr following NMDA treatment had rough, discontinuous plasma membranes, increased somatic volumes, cytoplasmic vacuoles, and a 'bath-chain' appearance in their axonal processes. In cortical neurons, adding 1 uM glycine only, with no NMDA, produced cultures that 24 hr later appeared healthy and morphologically similar to those observed before the treatment.  Experiments  where no NMDA was used showed a survival rate of 72.0±3.8% over the 24 hr period for cortical cultures.  3.4.3.2.Neuroprotection by (-)-B-cyclazocine in cortical neurons Cortical neurons were also protected from NMDA-induced neurotoxicity by (-)-pcyclazocine. This is illustrated in Figure 33, which shows a significant protecting effect by 1 uM (-)-p-cyclazocine (60.8±10.1% neuroprotection; n =7), and nearly full neuroprotection by 10 uM (-)-p-cyclazocine (93.4±17.2% neuroprotection; n = 7). With the latter concentration, the appearance of cells closely resembled that of cells not exposed to NMDA, indicating the neurotoxicity described by Olney et al. (1989) did not  -177occur. Raising the glycine concentration to 10 uM did not alter the effectiveness of (-)P-cyclazocine in cortical neurons (data not shown).  These results show that the  properties of (-)-p-cyclazocine to prevent neuronal damage were not restricted to the hippocampal subtype, and support a generalization to other neuronal subtypes susceptible to excitotoxic death mediated by NMDA receptors.  3.4.3.3.Effects of (+)-B-cyclazocine in cortical neurons The  (+)-enantiomer  of  p-cyclazocine, while  affording  some  degree  of  neuroprotection at higher conentrations, was not nearly as effective a neuroprotectant in vitro as the (-)-enantiomer in cortical neurons. Figure 33 shows the results with (+)P-cyclazocine:  1 pM (+)-P-cyclazocine produced 2.1 ±6.5% neuroprotection while 10  pM produced 51.3±9.9% neuroprotection.  The difference in neuroprotective potency  between the two enantiomers corresponds well with the difference in ICso's as determined in the electrophysiological experiments. This enantiomeric selectivity was also observed in hippocampal neurons, and suggests a similar mode of interaction with NMDA receptors in both cell types. 3.5.  Actions of the compounds on voltage-gated ion channels  3.5.1. Macroscopic currents in dorsal root ganglion (DRG) cell line neurons An ideal neuroprotectant would inhibit the neuronal process(es) responsible for producing excitotoxicity, while leaving elements involved in signal transmssion and maintenance of resting membrane potential intact. The latter functions are mediated by  -178Figure 34. TEA-sensitive delayed rectifier in D R G neurons. I was elicited by a K  sequence of 1800 ms depolarizing steps from the holding potential (-60 mV) to +60 mV in increments of +10 mV. (A) l recorded in control solution. (B) l in the same neuron K  K  (same protocol) but following extracellular application of 5 mM TEA.  -179-  A control  Figure 34  -180voltage-gated ion channels, chiefly N a and K* channels. +  In order to determine  possible interactions of (-)-p-cyclazocine and DM with voltage-gated ion channels, a study of whole-cell currents using specific voltage protocols to activate Na* and K* currents was carried out. For this series of experiments, a mouse DRG cell line was used since these neurons yielded sufficiently large whole-cell currents for study, whereas the cultured hippocampal and cortical neurons used in the previous studies were generally small. Robust TTX-sensitive sodium currents (l ) and TEA-sensitive Na  potassium currents (l ) were observed in the DRG neurons and were studied for their K  sensitivity to the presence of high concentrations of (-)-p-cyclazocine or DM. 3.5.1.1. Properties and pharmacology of I  K  In neurons, depolarizing steps from a holding potential of -60 mV activated an outward current whose size depended on the magnitude of the voltage step. Examination of the time course of this current revealed a fast approach to a non-zero steady-state level, as shown in Figure 34A. This current was sensitive to millimolar levels of externally applied TEA, as illustrated in Figure 34B. This current thus conforms to the properties of the delayed rectifier K* current and will subsequently be referred to as l . K  3.5.1.2. Effects of DM and (-)-0-cyclazocine on IK  The effects of DM and (-)-p-cyclazocine were tested at concentrations which completely  antagonized  NMDA  activity  in  the  electrophysiological  and  microspectrofluorimetric studies. The size of l« activated by the largest depolarizing step (to +60 mV from a holding potential of -60 mV) in controls was 473±91 pA (n = 8).  -181Figure 35. Neuronal l in the absence and presence of DM. (A) l in control solution, K  elicited by the same protocol as in Figure 34. extracellular application of 50 pM DM.  K  (B) l  K  in the same neuron after  Figure 35  -183For DM (50 pM), the current activated by this step was 86±23% of control (n = 6). An example of the action of DM against l is shown for a typical neuron in Figure 35. K  As well, (-)-p-cyclazocine (5 pM) did not act to alter l«; the size of the current activated by the step to +60 mV was 124±12% of control (n = 3). While it appears there was a small tendency for (-)-p-cyclazocine to actually increase the IK, this was due to the behaviour of a single neuron, in which the size of l increased by 45.7% K  upon superfusion of 5 pM (-)-p-cyclazocine. Factors such as changes in cell shape, affecting the voltage clamp, induced by solution flow may have contributed to thisaberrant result.  The above results show that neither DM nor (-)-p-cyclazocine  produce any actions against neuronal l at concentrations which fully inhibit NMDA K  activity and which provide strong neuroprotection against excitotoxic insult (Choi, 1987b).  3.5.1.3.Properties and pharmacology of In in neurons a  From a holding potential of -60 mV, depolarizing steps to -20 mV, preceded immediately by a hyperpolarizing prepulse (-100 mV) to remove sodium channel inactivation, induced an inward current (mean amplitude 3.6±0.8 nA) that inactivated within 8 ms and was blocked by low concentrations of TTX. This is shown in Figure 36, in which a typical neuron initially passing an inward current of 6.5 pA showed almost no current response to depolarization after exposure to 0.1 pM TTX. Full recovery of this current following TTX washout was observed in this neuron (not shown). This current conforms to previous descriptions of sodium channel activity in neurons (Hodgkin and Huxley, 1952; etc.) and will subsequently be referred to aS lNa-  -184Figure 36. TTX-sensitive sodium current in DRG neurons. l  Na  was elicited by a  15 ms depolarizing step to -20 mV following a 30 ms prepulse (-100 mV) from the holding potential (-60 mV). (A) l  Na  in control solution. (B) l  Na  in the same neuron (same  protocol) but following extracellular application of 0.1 pM TTX.  -185-  control  5 ms | 2 nA  B 0.1 / / M T T X  -20 mV mV  -100 mV  Figure 36  -186Figure 37. Neuronal l  Na  in the absence and presence of DM. (A) l  Figure 36 in the presence of control solution. (B) l extracellular application of 50 uM DM.  Na  Na  was elicited as in  in the same neuron but following  -187-  control  5 ms  B  2nA 50  \iM  DM  -20 mV  I  -60 mV  -100 mV  Figure 37  -1883.5.1.4.Effects of DM and (-)-B-cyclazocine onI  Na  Perfusion of the external solution with (-)-P-cyclazocine did not alter the size of l . Current amplitudes at the peak were 1.04±0.04 (n = 2) times as large as in control Na  when exposed to 5 pM (-)-p-cyclazocine (not shown), a concentration >30 times the ICso  for reduction of NMDA open channel probability, and a concentration which  abolished the [Ca ]i response to NMDA in the microspectrofluorimetric studies. This 2+  concentration of (-)-p-cyclazocine also did not alter the time course of l The addition of DM also did not affect the size of l I  Na  N a  N a  inactivation.  measured at the peak. The  in a typical neuron in control and in the presence of 50 pM DM is illustrated in  Figure 37. Current amplitudes in the presence of 50 pM DM, a concentration 10 times the ICso for reduction of NMDA P„ and which brought the NMDA-evoked [Ca ]j 2+  response to less than 5% of control, were 1.02±0.03 (n = 4) times the amplitude in control. The time course of inactivation was also unaffected by 50 pM DM. The  lack of effects  against  l  N a  by either  (-)-p-cyclazocine or DM  at  neuroprotective concentrations indicates that (-)-p-cyclazocine and DM would act as effective neuroprotective agents in neurons without compromising action potential generation or propagation.  3.5. J.5.Conclusions regarding drug effects on macroscopic currents in neurons  The addition of either DM or (-)-p-cyclazocine to the recording bath produced no changes in macroscopic l and l . The results for the compounds were determined at K  Na  concentrations that were at least 10 times the ICso's for blockade of NMDA currents,  -189and that were near the concentrations required to completely protect neurons from severe NMDA-type glutamatergic insult (Choi, 1987; Section 3.4). Since l« and l  Na  are  involved in both the generation and the termination of action potential spikes, the present results imply that neither agent would act to alter the time course or frequency of action potentials. It can therefore be predicted that the use of either DM or (-)-pcyclazocine in neuroprotectant therapy would produce minimal toxic side effects with respect to neuronal transmission.  3.5.2. Macroscopic currents in ventricular myocytes An agent used to protect the brain from excitotoxic damage would have maximal utitility if it affected no other organs, especially those organs, such as the heart, involved in life-sustaining processes.  Hence, voltage-gated currents in acutely  dissociated rat ventricular myocytes were studied as a indicator of the potential for (-)3-cyclazocine or DM to produce cardiac side effects. For this series of experiments, l and l  K  Na  were examined for their susceptibility to alteration by the two studied  compounds. As in the neurons, the agents were applied at concentrations well above the ICso's for inhibition of NMDA activity in hippocampal and cortical neurons, and that were near the concentrations shown to produce full neuroprotection in cultured neurons. 5.5.2. J.Properties and pharmacology of I in ventricular myocytes t0  Depolarizing steps to potentials more positive than -30 mV from a holding potential of -70 mV activated an outward current which quickly attained a peak and then inactivated to a non-zero steady-state level, as shown in Figure 38A. This current could be pharmacologically separated into two components, the first being a transient,  -190Figure 38. Actions of DM on cardiac l . Currents were activated with a depolarizing t0  step (+60 mV) from the holding potential (-70 mV). (A) l in control solution. (B) l in t0  the presence of 10 uM DM. (C) l in the presence of 50 uM DM. t0  t0  -191-  Control  B 10 \iM DM  6  c  ms  l 50 fiM DM  r  -20 mV  -70 mV -HO mV  Figure 38  2 n A  -192inactivating current sensitive to 4-AP, and the second being a slowly-activating residual current insensitive to 4-AP.  These two components are therefore considered as  separate entities distinguishable by their pharmacology and their kinetic behaviour (Apkon and Nerbonne, 1991; Castle and Slawsky, 1992; McLarnon and Xu, 1995), these being a rapidly inactivating component, which subsequently will be referred to as l , for transient outward current, and a residual delayed recitifier current. The present t0  study concentrated on the effects of DM and (-)-p-cyclazocine on two parameters of l : t0  the time course of inactivation (x) and the total charge passed by this entity. The effects of the agents on the residual steady-state portion of the current response was also studied and the results are presented below. 3.5.2.2.Effects of (-J-B-cyclazocine onI  t0  In control, voltage steps to +60 mV induced an outward current that decayed with a t of 44±3 ms (n = 6). The effect of (-)-fJ-cyclazocine on x was small, with a concentration of 10 pM bringing x to 80±11% (n = 3) of control. The integral of total charge movement, found by first subtracting the steady-state portion of the current and determining the area underneath the current amplitude vs. time curve, was also very weakly affected by the presence of (-)-p-cyclazocine. Values for total charge passed by l in the presence of (-)-p-cyclazocine were 75±18% (n = 3) of the value in control, t0  which was 87±14 nAms for n = 6 myocytes. The steady-state current remaining after inactivation of l slowly-activating l , such as the delayed rectifier. K  t0  may represent a  The size of this current in n = 6  cardiac myocytes was 0.97±0.21 nA in control solution.  Perfusion of 10 pM (-)-p-  -193cyclazocine had no effect on the size of this current, which was 102±15% (n = 3) of control following application of the agent. 3.5.2.3.Effects of DM on I  t0  In contrast to (-)-p-cyclazocine, DM had actions to decrease the l  t0  in cardiac  myocytes. These actions were concentration-dependent, with greater reductions in l  t0  being observed at 50 uM DM than at 10 uM DM. The decay of lu, was quickened by perfusion of DM; the value for T was 58±1% (n = 2) of control at 10 uM DM, and 38±20% (n = 2) at 50 uM DM (Figure 38B and C). The amount of charge passed was affected to an even greater degree, with 10 uM DM reducing the integral of charge movement to 27±6% (n = 2) of control, and 50 uM DM reducing it to 8±2% (n = 2) of control.  These actions on l  t0  would prolong the duration of the action potential in  cardiac myocytes and thus would generally represent an unwanted side effect at doses only slightly higher than those therapeutically effective for neuroprotection. The effect of DM on the delayed rectifier portion of the depolarization response was also studied. This entity was less sensitive to the presence of DM than l : no to  effect was observed at 10 uM DM (96±4% of control; n = 2), although a small but inconsistent effect was produced by 50 uM DM (63±20% of control; n = 2). The difference in susceptibility to DM between l  t0  and the residual steady-state current  indicates that, in the cardiac myocytes employed in the present study, the l delayed rectifier are pharmacologically distinguishable entities.  t o  and the  -194Figure 39. Actions of DM on cardiac l . Na  Currents were elicited by a 15 ms  depolarizing step to -20 mV following a 30 ms prepulse (-110 mV) from the holding potential (-70 mV); a series of 20 such activations is shown superimposed upon one another in each case. (A) l  Na  in control solution. (B) l  Na  in the presence of 10 uM DM.  Note the increase in block with each pulse, seen as a series of successively smaller peaks superimposed upon one another. (C) l  Na  in the presence of 50 uM DM.  -195-  Control  B 10 u M D M  40 ms  50 uM DM  +60 m V -70 mV  r  J  Figure 39  -1963.5.2.4. Properties and pharmacology qfI  Na  in ventricular myocytes  From a holding potential of -60 mV, a depolarizing step to -20 mV, delivered immediately after a hyperpolarizing prepulse (-90 mV) to remove sodium channel activation, elicited an inward current that attained a peak of -8.3±0.4 nA (n = 6) within 3 ms and quickly inactivated, within 6 ms, to zero net current (Figure 39A). While this pattern of activation and deactivation was similar to that observed in neurons, the pharmacology was different in that >10 times the concentration of TTX was required toblock this current in cardiac myocytes compared to neurons (not shown).  It was  therefore of interest to study the effects of the two compounds (-)-p-cyclazocine and DM in the myocyte preparation.  3.5.2.5. Effect of (-)-B-cyclazocine on I  Na  in ventricular myocytes  Perfusion of the myocytes with 10 pM (-)-p-cyclazocine did not alter the peak size of l , which was 108±2% of control (n = 2). No action to alter the time course of Na  inactivation was observed (not shown). The results of these experiments show the (-)P-cyclazocine did not block cardiac sodium channels at concentrations greater than those required for neuroprotection.  3.5.2.6. Effect of DM on Iv in ventricular myocytes a  Unlike (-)-p-cyclazocine, DM reduced the size of peak l  N a  in cardiac myocytes.  This action was observed without any effect to alter the time course of inactivation. The 1^ in a typical myocyte in control and in the presence of 10 and 50 pM DM .is shown in Figure 39. The reduction in the peak size was use-dependent, with a steadystate level of block being achieved by the 16th pulse if the pulse protocol was delivered  -197at a rate of 10 s" . This can be seen in the example illustrated in Figure 39B and C, 1  where succeeding pulses produced greater block and produced the thickening of the trace at the current peak. The use-dependent reduction of l  N a  suggests that DM inhibits  cardiac sodium channels via an open-channel block mechanism. Overall, the size of the peak l  N a  was 49±13% (n = 2) of control in the presence of 10 uM DM and 25±7% (n  = 2) of control in the presence of 50 uM DM. These concentrations are very near those producing similar levels of NMDA blockade in neurons and would therefore represent a potentially harmful cardiac side effect were DM to be used in neuroprotection therapy.  3.5.2.7.Conclusions regarding drug effects on macroscopic currents in ventricular myocytes  Perfusion of cardiac myocytes with a high concentration of (-)-p-cyclazocine produced no changes in l , the delayed rectifier, or l . These currents are involved in t0  Na  cardiac action potential generation and frequency and therefore any agent, such as (-)P-cyclazocine, which produces no effect in these currents at therapeutically relevant concentrations, would be expected to be relatively free of cardiac side effects. The action of DM to enhance inactivation of l , as represented by the decrease in x t0  following D M perfusion, and to decrease the amplitude of the delayed rectifier, indicates that cardiac K* channels are inhibited by this agent, even at a concentration of 10 uM which is close to the  IC50  for NMDA channel blockade (5 pM).  This  concentration also produced use-dependent open-channel block of cardiac Na* channels. The above actions of DM would interfere with the process of action potential generation and termination and would alter spike frequency. It is therefore a legitimate  -198concern that concentrations of DM effective in neuroprotection could potentially produce cardiac side effects, such as arrhythmias.  -199-  DISCUSSION  4.  4.1.  Neuroprotection by (-)-p-cyclazocine 4.1.1. NMDA  ion channel  blockade  by (-)-B-cyclazocine  results in  neuroprotection One important finding of the work described in this thesis is that the neuroprotectant properties of (-)-p-cyclazocine are based on blockage of NMDA ion channels. This results from the agent's effects to decrease the open state probability (P ) of single NMDA ion channels in the electrophysiological studies and its action to 0  inhibit  intracellular  free  calcium  ([Ca ]i) 2+  responses  to  NMDA  in  the  microspectrofluorimetric studies. The antagonism of NMDA ion channels by this agent was a strong, if not complete, determinant of the neuroprotective effect: (-)-pcyclazocine was, on one hand, a potent neuroprotectant while, on the other hand, acting as a potent non-competitive NMDA antagonist with little or no activity on other ion channels. In particular, the fura-2 measurements indicated little or no effects of the compound on AMPA or kainate responses or on responses mediated by voltage-gated Ca  2 +  channels; whole-cell patch clamp measurements showed the compound to have  no effect on voltage-gated N a or K* channels. +  The potent neuroprotective properties of (-)-p-cydazocine were mirrored in its potent blockade of responses to applied NMDA in both hippocampal and cortical neurons. In hippocampal neurons, the  ICso  for (-)-p-cyclazocine reduction of P was 84  nM in patch-clamp measurements of single NMDA ion channels, whereas the  0  ICso  for  attenuation of NMDA-evoked increases in intracellular free calcium was 270 nM. The  -200three-fold difference in IC^, values may represent differences in the parameters being measured by the two techniques: the microspectrofluorimetric method measures drug effects on rises in intracellular free calcium, whereas the  electrophysiological  measurements reflect drug actions on both sodium and calcium influxes through the NMDA-associated ion channel. In the cortical neurons, the  IC50  for reduction of single  NMDA ion channel open state probability was 680 nM, whereas the  IC50  for inhibition of  NMDA-evoked intracellular free calcium responses was 220 nM. Again, the differences may be referable to the methodology employed in the measurements. 4.1.2. Actions of (-)-3-cyclazocine relevant to neuroprotection The study of the interaction of (-)-p-cyclazocine with single NMDA ion channels revealed two main effects of the compound on the kinetic behaviour of the channel. First, the mean open time of the channel was reduced, in a concentration-dependent manner, by (-)-p-cyclazocine. This reduction was observed in both hippocampal and cortical neurons. The action of (-)-p-cyclazocine to reduce channels mean open time (Up) is a feature that would contribute to its antagonism of NMDA receptor-ion channel complexes independently of the channel opening frequency, and hence P . 0  Diminution of the frequency of NMDA-activated unitary currents was observed concomitantly with the decrease in t o following application of (-)-p-cyclazocine. These P  two effects of the compound combined to produce a decrease in the open state probability of the ion channel, such that the P was 50% of control at (-)-p-cyclazocine 0  concentrations of 84 nM and 270 nM in hippocampal and cortical neurons, respectively. Since macroscopic current can be expressed as the product of unitary current, the number of conducting channels, and P , it follows that changes in P can be used as a 0  0  -201comparison with other agents' effects on whole-cell currents. The IC50 values obtained for (-)-p-cyclazocine show this compound to be one of the most potent blockers of NMDA activity known, with similar potency as P C P . Indeed, only dizocilpine (MK-801) is a more potent uncompetitive  NMDA antagonist (Huettner  and Bean, 1988;  MacDonald et al., 1991). 4.1.2.1. Open channel block and the on-rate constant  A sequential open channel block model was used to characterize the interaction of (-)-p-cyclazocine with the NMDA ion channel. Plots of the inverse of mean open time versus (-)-p-cydazocine concentration were linear for both hippocampal and cortical neurons, consistent with a simple open channel blockade scheme.  This finding is  important, since it indicates that, over the concentration range studied, (-)-pcyclazocine acts only as an uncompetitive channel blocker.  Such a relationship is  characteristic of a bimolecular interaction between the blocking agent and a binding site on the receptor-ion channel complex. The blocking rate constants, k , obtained 2  from the slope of this plot, were found to be 7.6 x 10 M'V and 1.2 x 10 M'V at V = 8  7  p  40 mV in hippocampal and cortical neurons, respectively.  Open channel block has  been used previously to characterize the interactions of a number of agents, including dizocilpine, P C P and several tricyclic antidepressants, with the NMDA channel (Huettner and Bean, 1988; Sernagor et al., 1989; MacDonald et al., 1991). The results from whole-cell patch-clamp studies using ketamine, PCP, dizocilpine, and memantine have provided estimates for onward rate constants in the range 2 x 10 - 3 x 10 M'V 4  (MacDonald et al., 1991; Chen et al., 1992).  5  A s noted previously for dizocilpine  -202(Huettner and Bean, 1988), the blocker can gain access to its site only when the channels are open (or, more precisely, blockade via open channels proceeds at a much faster rate than via closed channels; Kloog ef a/., 1988).  Therefore, whole-cell  measurements of blocking rate constants must be corrected for the low open probability of the NMDA channel. Estimates of P with recordings of unitary currents from outside0  out patches have been given at 0.006 for 2 pM NMDA in outside-out patches (Bertolino et al., 1988) and 0.026 for 30 pM NMDA in cell-attached patches (McLarnon and Sawyer, 1993); these values for P indicate the channel was open 0.6% and 2.6% of 0  the time, respectively. In the present study, the P was 0.08 in hippocampal neurons 0  and 0.05 in cortical neurons for 25 uM NMDA at V = -40 mV. Thus, using a P value of p  0  0.05, corrected whole-cell onward rate constants would generally fall in the range 5 x 10 - 3 x 10 M'V . The cluster of onward rate constants in the range 10 - 10 M" s'  1  suggests a similar site of action for the various agents. Furthermore, values near 10  7  s  7  1  7  8  1  M'V are characteristic of blockers for other channel systems, such as with blockade of 1  acetylcholine receptors by the lidocaine derivative QX-222, and are suggestive of a diffusion-limited process (Neher and Steinbach, 1978). The high values of the on-rate constants found for (-)-p-cyclazocine in the present study are reminiscent of the smaller amantadine derivatives investigated by Antonov and co-workers, suggesting that (-)-pcyclazocine resembles these agents in terms of a possible deep binding site in the channel vestibule (Antonov etal., 1995; Antonov and Johnson, 1996).  -2034.1.2.2.The off-rate constant  Distributions for channel closed time were altered in the presence of (-)-pcyclazocine. Sections 3.2.6.2 and 3.2.12.2 describe the presence of a long closed time component seen after (-)-P-cyclazocine addition.  In theory, the unblocking rate  constant can be found from comparison of closed time distributions in control solution and in the presence of the blocking compound. In the present experiments, a clear differentiation between channel blocking transitions to a non-conducting state and normal transitions to the closed state was not possible. For example, no evident rapid transitions (channel flickering) were observed in the presence of any of the studied compounds, including (-)-p-cyclazocine. However, closed time distributions with (-)-pcyclazocine showed a longer, third time component which was generally absent, or rarely present, in control closed time distributions.  The change in the longest time  component most likely represents the time the drug molecule associates with the NMDA channel. The presence of a time component in the 200 - 400 ms range was the clearest effect of (-)-p-cyclazocine on closed time distributions. With the assumption that this long time component was a measure of the observed action of (-)-pcyclazocine to significantly reduce open frequency, the unblocking rate constant was estimated to be near 5 s' . Thus, channel block by (-)-p-cyclazocine was associated 1  with a relatively slow off-rate constant, compared with M g , for drug binding to a 2+  channel site. This was consistent with the lack of channel flickering observed in the presence of (-)-p-cyclazocine at any patch potential or drug concentration.  -2044.1.2. ^.Potency of (-)-B-cyclazocine and comparison with other agents  The ICso values obtained for (-)-p-cyclazocine blockade of NMDA ion channels in the present study can be compared with the respective values of 1.2 pM and 10 nM obtained for memantine (Chen et al., 1992) and dizocilpine (Huettner & Bean, 1988). In the case of the latter compound, its low ICso has been attributed to a low k. , or, in 2  other words, its protracted association time with the channel site. Indeed, as pointed out above, our results suggest that the different magnitudes of ICso are primarily dependent on differing values of k. for blockade of the NMDA channel, since onward 2  blocking rate constants are not markedly different among agents. The present results indicate that (-)-p-cyclazocine dissociates from its blocking site much faster than dizocilpine, which appears to remain associated with its site for >10 s (Huettner and Bean, 1988; Parsons etal., 1995). This point is important because the unusually slow offset time of dizocilpine is suggested to be a limiting factor in the clinical utility of this anticonvulsant (Chen et al., 1992; Lipton, 1993).  For example, dizocilpine has been  found to impair motor function in intact rats (Rogawski et al., 1991; Parsons et al., 1995). It would appear that the potency of a given agent is proportional to the amount of time spent by the agent in the channel blocking site; hence, the magnitude of k is a 2  major determinant in the blocking potency of uncompetitive antagonists.  4.1.2.4.Inhibition of NMDA-evoked [Co *]/ responses 2  Fluorescence microscopy of both hippocampal and cortical neurons loaded with the free calcium marker fura-2 revealed (-)-p-cyclazocine to be a potent antagonist of NMDA-evoked [Ca ]j responses. In these experiments, the level of (-)-p-cyclazocine 2+  -205blockade increased each time the agonist was applied until an equilibrium level of blockade was achieved. Conversely, during washout, each agonist application in the absence of (-)-p-cyclazocine resulted in a greater change in [Ca ]j than with the 2+  previous application.  The marked use-dependence observed with (-)-p-cyclazocine  blockade has been previously noted for NMDA responses in rat neocortical wedges (Church et al., 1991) and is qualitatively similar to that seen with PCP, ketamine, and dizocilpine in macroscopic currents recorded from cultured murine hippocampal neurons following application of NMDA, L-aspartate, or L-glutamate (MacDonald et al., 1987; MacDonald et al., 1991). The degree of use-dependence (that is, how many agonist applications are required to achieve steady-state blockade or drug wash-off) is a consequence of the speed of interaction of a given agent with the blocking site, which is in turn governed by the blocking rate constant k and the unblocking rate constant k. 2  2  . Indeed, of all the agents tested, only (-)-p-cyclazocine exhibits any degree of use-  dependence, as evidenced by the fura-2 measurements. Since this compound showed the slowest off-rate [i.e., the lowest k. ), it is likely that the degree of use-dependence 2  observed with a given compound is mainly dependent on the magnitude of k. . 2  4.1.2.5. Trapping of antagonist within closed channels  Several bolus applications of NMDA were necessary in order to attain a new steady-state response following discontinuation of (-)-p-cyclazocine superfusion. This suggests the agent was trapped within the channel due to channel closure following anatagonist binding.  Alternatively, this suggests that (-)-p-cyclazocine does not  prevent channel closure; this aspect of the compound's action is supported by thke  -206similarity between the microscopic K found in the single-channel experiments and the D  whole-cell Ko determined in the fura-loaded neurons (Antonov et al., 1995). The size of the off-rate constant may be related to the tendency for the agent to be trapped within the ion channel. In the case of (-)-p-cyclazocine, the magnitude of k. found for (-)-P2  cyclazocine in the electrophysiological studies was such that the length of time the drug molecules were associated with their site exceeded the normal opening/closing kinetics of the channel. Since (-)-p-cyclazocine exhibited the smallest k. , the requirement of a 2  number of agonist activations in order to wash away (-)-p-cyclazocine following its removal from the superfusion medium appears to be inversely correlated with the size of k. . 2  This facet of (-)-p-cyclazocine's actions on NMDA activity is consistent with a  "drug-trapping" scheme in which the channel can close with the antagonist molecule(s) still remaining within the channel. Egress of the agent from closed channels proceeds at such a slow rate that it may be a requirement that the channel must be open for the blocker molecule to exit. In this sense, the model of (-)-p-cyclazocine interaction with NMDA ion channels is consistent with the "guarded receptor" hypothesis originally proposed by Starmer and colleagues (1986) for local anaesthetic block of Na* channels, and modified by MacDonald et al. (1991) for uncompetitive antagonist block of NMDA channels, where access of a channel blocker to its site is controlled by the channel gating mechanism.  4.1.2.6.Potent neuroprotection with B-cyclazocine is stereospecific The compound (-)-p-cyclazocine was found to be highly potent in the present neuroprotection model involving NMDA-induced excitotoxicity, with a high level of  -207neuroprotection being achieved at a concentration of 1 uM. cyclazocine action against neuronal C a  2 +  The lack of  (-)-P-  channels, neuronal voltage-activated Na* and  K* channels, and cardiac voltage-activated N a and K* channels suggests that the +  neuroprotective effect of (-)-p-cyclazocine was chiefly due to its action to block NMDA ion channels. Whereas other agents which have been shown to be uncompetitive antagonists of the NMDA receptor-ion channel complex have been also found to possess neuroprotective properties (Olney et al., 1986; Choi, 1987b; Rogawski et al., 1991; Chen et al., 1992), to date, only MK-801 has been shown to be more potent than (-)-P-cyclazocine against NMDA-mediated neurotoxicity (Lysko  etal., 1989).  The actions of p-cyclazocine on NMDA ion channels were stereospecific, as evident from the differential potency between the (-)- and (+)-enantiomers in their block of NMDA unitary currents, their inhibition of intracellular free calcium and their protection of cells subjected to excitotoxic insult. Stereospecifity of p-cyclazocine, with the (-)-enantiomer being more potent than the (+)-enantiomer, has previously been observed in NMDA responses of rat cortical wedge preparations in vitro and of rat spinal neurons in vivo (Church et al., 1991) and in binding experiments assaying the competition of the two enantiomers for a radiolabeled P C P receptor ligand (Todd et al., 1990). In PCP-trained rats, (-)-p-cyclazocine was 50 times more potent in substituting for P C P than (+)-p-cyclazocine (Slifer and Balster, 1988). The higher potency of the ()-enantiomer of p-cyclazocine is consistent with the actions of other benzomorphan compounds on NMDA receptors, including pentazocine and a-cyclazocine (see Church and Lodge, 1990; an exception is SKF 10,047), and is opposite to compounds from  -208other classes acting on this site, including ketamine (Lodge et al., 1982) and the morphinans (Church etal., 1985). 4.1.2.7.Non-opiate mechanism  The use of outside-out patches would preclude indirect actions, such as effects via opioid receptors, of (-)-(3-cyclazocine on currents through the NMDA ion channel. Since the potencies of (-)-p-cyclazocine in the microfluorescence studies and in the neuroprotection studies were similar to the potency in the electrophysiological studies, the possible contribution of opioid receptors to drug effects was likely small.  For  example, both morphine and naloxone have been previously found to each have no effect on NMDA-induced [Ca ]i in hippocampal neurons (Church et ai, 1994a). While 2+  opiates have been shown to protect neurons from NMDA-mediated cell death in vitro (Choi and Viseskul, 1988), high concentrations of the agents were required for neuroprotection, including the classical p opioid receptor agonist morphine (40 - 60% neuronal injury reduction at [morphine] = 100 pM); hence, the opiates were far less potent than (-)-p-cyclazocine was found to be in the present study.  Finally, the  anxiolytic actions of (-)-p-cyclazocine were found to be PCP-like rather than morphinelike in a punished behaviour model in rats (Porter et ai, 1989).  Nevertheless, the  possibility that opioid actions of (-)-p-cyclazocine may have played a small role, or even may have produced a combined effect with its NMDA-blocking properties, in its neuroprotective capacity cannot be excluded and remains to be investigated in the future.  -2094.1.2.8.Predictive power of model  The application of an open channel block model to the results of the electrophysiological studies, subsequently confirmed by the observation of usedependence in the microspectrofluorimetric experiments, was successfully used to predict the neuroprotective property of (-)-p-cyclazocine. Its high potency in reducing NMDA open channel probability in outside-out patches and in reducing NMDA-evoked [Ca ]i 2+  responses was matched by its high potency in protecting neurons from damage  induced by exposure to a high concentration of NMDA.  The relationship between  electrophysiological potency (and potency in the microspectrofluorimetric studies) held true for two types of neurons, hippocampal and cortical.  Furthermore, the rate  constants associated with blockade also are of predictive value, since it was found that potency of a given agent to reduce NMDA open channel probability, and hence to protect against NMDA receptor-mediated neuronal damage, is a consequence of the magnitude of the unblocking rate constant, k. . 2  Lipton (1993) has proposed a k.  2  "window" within which an NMDA antagonist is effective and safe. Agents with high k.  2  values, such as with the fast channel blocker M g , interact too quickly with the ion 2+  channel and produce little overall antagonism.  On the other hand, compounds with  extremely low k. values, such as MK-801, remain associated with the ion channel for 2  long periods of time and consequently produce toxicity due to interference with the processes in which the NMDA receptor-ion channel play an important functional role, e.g., synaptic transmission.  The k.  2  values found for (-)-p-cyclazocine  with  hippocampal and cortical neurons in the present study is much smaller than the k_  2  value for M g  2+  (as evidenced by the observed lack of flickering between open and  -210blocked states in the presence of (-)-p-cyclazocine) yet greater than the k. for MK-801 2  (around 0.003 s" ; MacDonald et al., 1991), thus falling into the "window" of acceptable 1  k- values for effective and well tolerated neuroprotectants. 2  It is therefore suggested  that the strategy described in the present work is an accurate identifier of putative neuroprotectants, and that further investigations using these techniques could be employed  in order  to  identify  other  uncompetitive  NMDA  antagonists  with  neuroprotective properties. 4.1.2.9. Selectivity for NMDA channels vs. other channels  Challenges with other excitants (i.e., kainate and AMPA) revealed (-)-pcyclazocine to be a selective blocker of NMDA ion channels vs. non-NMDA ion channels. The selectivity of (-)-p-cyclazocine for NMDA over non-NMDA ion channels agrees  with  the  findings  microelectrophoretically  of  applied  Church NMDA,  et but  al.  (1991)  that  not the AMPA  responses  receptor  to  agonist  quisqualate, were inhibited when the agents were used with rat cortical wedges in vitro and with rat lumbar spinal neurons in vivo. The selective actions of (-)-p-cyclazocine on NMDA ion channels are consistent with the NMDA-selective effects of other benzomorphans, including /V-allylnormetazocine (SKF-10,047; Berry et al., 1984), a cyclazocine (Thomson and Lodge, 1985; Church and Lodge, 1990), and pentazocine (Church and Lodge, 1990). Raised rC-evoked [Ca ]i responses were unaffected by (-)-p-cyclazocine in both 2+  hippocampal and cortical neurons.  This is in contrast with other  antagonists, such as DM (see below) and P C P , which antagonize C a  uncompetitive 2 +  channels at  -211concentrations only slightly above those effective in blocking NMDA responses (ffrench-Mullen and Rogawski, 1992).  The responses to raised K* in the present  experiments were due chiefly to C a  influx mediated through high voltage-gated  2+  calcium channels activated following neuronal membrane depolarization produced by the K* applications.  Raised K*-evoked  [Ca ]i 2+  responses in cultured hippocampal  neurons have been shown to be chiefly mediated via dihydropyridine-sensitive ion channels, or L-type C a [Ca ]i 2+  2+  channels (Church et al., 1994a); however, a small part of the  response to raised K* was contributed by a m-conotoxin-sensitive component  (most likely N-type C a  2 +  channels) and by a dihydropyridine- and co-conotoxin GVIA-  resistant component. These experiments with the fura-2-loaded cells constitute the first evidence that (-)-p-cyclazocine is unable to block voltage-activated calcium channels, whether they be L-type, N-type, or non-L,N-type, in CNS neurons at concentrations which are completely effective in attenuating NMDA activity. No effect of (-)-p-cyclazocine on voltage-gated ion channels were found in whole-cell patch clamp studies on neurons.  Currents through voltage-gated N a  +  channels, which are responsible for the rising phase of the action potential, were not altered by the presence of (-)-p-cyclazocine, at a concentration sufficient to block NMDA activity by >90% (5 uM). The tetraethylammonium (TEA)-sensitive l  Kl  a delayed  rectifier-type K* current which acts to repolarize neurons following action potential spikes, was also not affected by the same high concentration of (-)-p-cyclazocine. The lack of effects on amplitudes and kinetic behaviour of voltage-gated N a and K* +  currents would suggest the compound would not impair neural transmission at concentrations which are neuroprotective due to NMDA blockade. This point does not  -212include the possibility that (-)-p-cyclazocine could act on other K* channels which contribute to the repolarization phase of action potentials. High concentrations of (-)-p-cyclazocine (5 pM) did not alter voltage-gated ion currents in ventricular myocytes. Since the pharmacology of l  Na  in cardiac myocytes  differs somwhat from that in neurons (the most salient example being the ten-fold difference in potencies of TTX blockade), it was reasonable to check for a differential action of (-)-p-cyclazocine on N a channels from the two tissue types. However, (-)-p+  cyclazocine was found to be without effect on cardiac l . The two components of the Na  rC-carried current, the initial l phase and the residual delayed-rectifier l , were also t0  K  unaffected by a high concentration of (-)-p-cyclazocine which was fully effective in blocking NMDA activity in neurons. These results show the pharmacology of (-)-Pcyclazocine to be similar in two types of excitable tissue, and also suggests that the use of (-)-p-cyclazocine as a neuroprotectant would not be expected to cause cardiac side effects, such as ventricular arrhythmias.  4.1.3. (-)-B-cyclazocine is a candidate neuroprotectant The rescue of hippocampal neurons from NMDA-induced cell death by low concentrations of (-)-p-cyclazocine suggests further study with respect to its potential as a neuroprotectant compound. neuropathologies,  including  The involvement of NMDA receptors in several  epilepsy, neurodegeneration  following  episodes of  ischaemia, hypoxia, hypoglycaemia, or CNS trauma, and degenerative disorders such as Huntington's chorea, Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis, has been targetted as a possible focus for therapeutic intervention (Choi, 1988a; Choi and Rothman, 1990). The demonstrated uncompetitive nature of (-  -213)-P-cyclazocine antagonism of NMDA activity in hippocampal neurons suggests that drug efficacy would be enhanced with escalating levels of glutamate.  This point is  important because drug effects on synaptic transmission mediated by normal levels of glutamate would be minimized, whereas NMDA receptors exposed to relatively high glutamate concentrations, the presence of which may be central in precipitating neurodegenerative  disorders (Rogawski, 1993), would be selectively  targetted.  Furthermore, the higher magnitude of the unblocking rate constant compared to the clinically intolerable MK-801 suggests that the application of (-)-p-cyclazocine in C N S therapy would be relatively unhindered by neurological side effects.  Therefore, the  results obtained in the present study are indicative of (-)-p-cyclazocine's candidacy as a clinical agent appropriate in the therapeutic intervention of the pathology associated with stroke and chronic neurodegenerative disease. 4.2.  NMDA antagonism by DM  4.2.1 Interactions of neuroprotectant concentrations of DM with NMDA ion channels DM has been previously shown to act as an NMDA blocker (Church et al., 1985; Church et al., 1991b) and as a protector against glutamate insults in vitro (Choi, 1987b) and against ischaemic conditions in vivo (Steinberg et al., 1988). At present, however, the interactions of DM with the NMDA receptor-ion channel are not well documented. W e have used measurements of DM actions on unitary NMDA currents and NMDAevoked [Ca ]j responses to address this point. 2+  -214-  4.2.2. Actions of DM relevant to neuroprotection 4.2.2.1. Alteration in NMDA ion channel kinetics  The actions of DM on unitary currents activated by NMDA included reversible, concentration-dependent reductions in mean open time and in opening frequency. Together, these changes resulted in decreases in the P of the NMDA-associated ion 0  channel. Similar alterations in unitary NMDA kinetics were seen with (-)-p-cyclazocine, suggesting that the two agents share a common mechanism of action with respect to NMDA antagonism. The observation of the reduction in P in isolated patches shows 0  the alteration in behaviour of NMDA ion channels to be a direct effect of DM, rather than its metabolite dextrorphan, to which DM is converted in vivo and which has a higher affinity as an NMDA antagonist (see Church etal, 1985). 4.2.2.2. Open channel block by DM and the forward rate constant  DM, at concentrations of 5 to 50 pM, had no effect on the magnitudes of unitary currents induced by activation of the 50 pS primary conductance state of the NMDA channel. This result, coupled with this agent's action to diminish channel mean open times and the frequency of openings, was suggestive of open channel block by DM. Previous studies (see below) have used various open channel block schemes to analyze drug interactions with the NMDA channel. In this work, a simple uncompetitive open channel block model appeared adequate to describe DM's actions on unitary NMDA currents, since a plot of the inverse of mean open time, in the presence of DM, vs. drug concentration was linear. The blocking rate constant k was obtained from the 2  slope of the relation and had magnitudes of 8.5 x 10 M'V 6  1  in hippocampal neurons  -215and 7.9 x 10 M'V 6  1  in cortical neurons; these on-rates were similar, although slightly  slower, than those for (-)-p-cyclazocine (7.6 x 10 M' s" in hippocampal neurons and 8  1  1  1.2 x 10 M'V in cortical neurons) and not far removed from the blocking rate constant 7  1  of 3 x 10 M'V that has been estimated for dizocilpine block of unitary NMDA currents 7  1  (Huettner and Bean, 1988). Thus, it would seem that onward (blocking) rate constants for a range of putative uncompetitive NMDA channel blocking drugs are not markedly different. 4.2.2.3. Voltage-dependence of on-rate constant  The on-rate constant k was found to depend on potential, using the single2  channel methods employed in the present study. The values for k increased with more 2  negative V ; however, this voltage dependence was less than that associated with M g  2+  p  block, where the voltage-dependence was quite pronounced (Huettner and Bean, 1988). It should also be noted that mean open times in controls were dependent on potential and diminished by about 20% for each 20 mV increase in patch hyperpolarization. The voltage dependence associated with channel blockade is thus strongly influenced by an additional contribution from the voltage dependence of the unblocking rate constant, as evidenced by the trend for greater reduction in channel opening frequency with more negative V . Voltage-dependence for off-rate constants p  has been observed in previous studies with dizocilpine (Huettner and Bean, 1988); voltage-dependence in the onward rate constant has also been noted with several derivatives of adamantane (Antonov et al., 1995; Antonov and Johnson, 1996), of  -216which memantine and adamantane are also derivatives showing voltage-dependent onand off-rate constants (Blanpied et al., 1997). 4.2.2.4. High off-rate constant for DM and the implication for clinical use  It would seem evident that differences in potencies for uncompetitive NMDA channel-blocking agents primarily reflect differences in the magnitudes of the unblocking (off-rate) constants from a site(s) associated with the NMDA channel. In this regard, DM was found to wash off relatively rapidly after application of control solution to the excised patches and exhibited similar kinetic behavior of unitary currents to that observed with memantine (Chen et al., 1992).  Despite having relatively fast  kinetics of interaction with the NMDA receptor-operated channel, DM appears to possess a narrow therapeutic window in seizure tests on rats (Loscher and Honack, 1993), although it has been employed successfully for treating seizures in humans (e.g., Schmitt et al., 1993) and exhibits little toxicity in clinical dose-escalation safety studies (Steinberg and Bell, 1992).  The former findings may reflect DM's rapid  metabolism in vivo to the relatively high-affinity uncompetitive NMDA antagonist dextrorphan (see Church et al., 1989) and does not, therefore, mitigate the suggestion that relatively low-affinity uncompetitive NMDA antagonists with fast kinetics of interaction with the NMDA receptor-operated channel may be well-tolerated clinically.  4.2.2.5. The nature of the DM binding site: biophysical studies  Biophysical studies with DM were carried out in order to obtain further information about the nature of the binding site for uncompetitive blockers and the manner in which the blockers interact with the site.  An outside-out path exposed  -217simultaneously to DM and the endogenous uncompetitive NMDA antagonist M g behaved as if the two agents were acting at different sites, with M g access of DM to its site.  2+  2+  possibly reducing  The results of temperature studies on cortical neurons  intended to elucidate the nature of the interaction of DM with NMDA ion channels were inconclusive. with a Q  1 0  While the conductance mediated by NMDA channels was associated  of 1.4, indicating a process rate-limited by diffusion, channel kinetics were  associated with much higher Qio's, suggestive of a process more complicated than one governed  by free  diffusion  of  channel elements  between  two  locations  or  configurations. This high temperature dependence in itself served to mask the effects of temperature on DM access to and egress from its blocking site. Therefore, because of the complex nature of NMDA ion channel kinetics, these experiments were not able to yield any definitive information regarding the manner in which DM inhibits current flow through the channel. 4.2.2.6.DM reduction of NMDA-evoked [Ca J 2+  i  responses  Consistent with its reduction of unitary NMDA P in outside-out patches, DM 0  concentration-dependently and reversibly inhibited NMDA-evoked [Ca ]i increases in 2+  both hippocampal and cortical neurons. agreement between the  IC50  With hippocampal neurons, reasonable  value obtained in the microspectrofluorimetric studies and  the DM concentration associated with a channel P of 0.5 (4 uM in both cases) was 0  found.  Similarly, with cortical neurons, the DM concentration at which standardized  NMDA P was 0.5, 4 uM, matched closely the 0  NMDA-evoked [Ca ]i increases (5 uM). 2+  IC50  value obtained from inhibition of  These values show that the sites through  -218which uncompetitive antagonism of NMDA activity is effected in the two neuronal subtypes is indistinguishable by DM. There was no use-dependence associated with DM blockade of NMDA-evoked  [Ca ]i 2+  increases, with equilibrium blockade being  reached after a single excitation. This is contrast to (-)-p-cyclazocine, for which up to 6 NMDA applications were necessary to achieve steady state. DM was also «20X less potent than (-)-p-cyclazocine in inhibiting NMDA responses.  Together, these two  findings suggest that the potency of an agent as an open channel blocker is related to its off rate constant, with the faster approach of DM to equilibrium blockade being :  indicative of a higher k_. 2  4.2.2.7.DM actions on voltage-activated Ca * channels 2  The lack of selectivity of DM for NMDA-activated ion channels vs. voltageactivated C a  2 +  channels was observed in both hippocampal and cortical neurons. The  action of DM on C a  2 +  channels is consistent with previous observations in cultured  hippocampal neurons (Church et a/., 1991b), in synaptosomes from whole rat brain (Carpenter et al., 1988), and in PC12 cells (Carpenter et al., 1988) and has been observed to occur concurrently with NMDA antagonism, albeit with lesser potency (Jaffe er al., 1989; Church et al., 1991b).  It has been suggested that the  neuroprotectant action of DM (Choi, 1987b; Steinberg etal., 1988; Tortella etal., 1989) may be related to the combination of its actions at the two sites; although C a  2 +  channel  antagonism has shown promise as a neuroprotective strategy (Siesjo, 1986; Pizzi etal., 1991), problems related to efficacy have been encountered (Koroshetz and Moskowitz, 1996).  -2194.2.2.8. Lack of effect on non-NMDA ion channels  The actions of DM to reduce responses evoked by kainate and A M P A in hippocampal neurons was no larger than the reduction in K*-evoked C a  2 +  responses  observed simultaneously. Therefore, it is likely that DM acted to reduce the kainateand AMPA-evoked [Ca ]i responses by virtue of its effect to block C a 2+  mediated  the  depolarization.  [Ca ]i 2+  increase  produced  by  non-NMDA  2 +  channels that  receptor-mediated  Interestingly, in cortical neurons, DM had no effect on kainate- and  AMPA-evoked [Ca ]j responses at a concentration that resulted in C a 2+  2 +  channel  blockade. It would appear that, in cortical neurons, [Ca ] responses to kainate and 2+  :  A M P A were not dependent on activation of either NMDA or C a  2 +  channels that were  sensitive to DM. One tempting explanation for this phenomenon would be that these cortical neurons contained non-NMDA receptors that were permeable to calcium, as with AMPA receptors lacking the GluR2 subunit (McBain and Mayer, 1994; Mody and MacDonald, 1995); further studies are required to clarify this issue.  4.2.2.9. Lack of DM actions on voltage-gated ion channels in neurons and cardiac myocytes  Very little work has been done with regard to DM actions against voltageactivated ion channels. This is surprising, because DM has entered clinical trials for neuroprotection (Koroshetz and Moskowitz, 1996), using doses higher than required for its antitussive action. The purpose of this section was to investigate the effects of DM on voltage-gated K* and N a currents using whole-cell studies in neurons. +  These  experiments showed that, like (-)-p-cyclazocine, DM is ineffective against other voltage-  -220gated ion channels.  Neither the TTX-sensitive l  Na  .nor the TEA-sensitive l  K  were  blocked by DM, even at a concentration of 50 uM, 10X the IC o for inhibition of NMDA 5  open channel probability.  Two conclusions can be made from these observations.  First, effects of DM to impair intraneural transmission would not be expected. Second, together with the results with (-)-p-cyclazocine on the same neurons, the lack of effect of DM on voltage-activated N a and K* channels shows that neither of these two +  channel types played a significant role in the neuronal damage produced by exposure to a high concentration of NMDA, and that the neuroprotective properties of DM (Choi, 1987; Steinberg et al., 1988; Tortella et al., 1989) are not dependent on N a or K* +  channel blockade. In contrast to the results obtained in neurons, studies into the effects of DM on whole-cell currents in ventricular myocytes revealed blockade of cardiac voltage-gated ion channels. The two distinguishable components, l  t0  and the delayed K* current, of  the outward K* response to depolarizing pulses were reduced relative to control in the presence of DM. Also, DM concentration- and use-dependently reduced the size of the N a current, suggesting an open-channel block of cardiac N a channels. It is possible +  +  that DM may have acted to shift the voltage-dependence of activation of the N a  +  current; more detailed experiments would be necessary in order to elucidate the nature of DM interaction with cardiac N a channels. The observation that DM blocks voltage+  gated ion channels in cardiac tissue without a parallel effect in neuronal tissue suggests that both N a and K* channels show variation between the two tissue types in +  terms of pharmacological properties.  The introduction  of DM for use as a  neuroprotective agent should, therefore, proceed with considerable caution, as effects  -221on cardiac voltage-gated ion channels were observed at concentrations not much higher than the IC50 for NMDA antagonism, the effect of DM most relevant to its neuroprotective action. 4.3.  NMDA antagonism by L-687,384 4.3.1. Interaction of L-687,384  with NMDA ion channels  4.3. 1. 1. Open-channel block of NMDA ion channels  The actions of the a ligand L-687,384 on NMDA channel kinetics differed from the other tested agents in that no change in opening frequency was observed. However, similar to (-)-p-cyclazocine and DM, L-687,384 concentration-dependently reduced mean open time of the NMDA ion channel, and hence showed an effect to reduce channel open probability at micromolar concentrations.  The increase in Up"  1  was found to be linear with increasing [L-687,384], consistent with an open channel block model for this compound.  Therefore, despite its lack of effect on opening  frequency, L-687,384 likely antagonized NMDA activity via the same open channel block mechanism as (-)-p-cyclazocine and DM.  4.3.1.2.Fast kinetics of block  The inability of L-687,384 to alter channel opening frequency at concentrations up to 40 uM was reflected in the closed time distributions, which also were unchanged by the compound.  The failure to observe an additional component in closed time  distributions may have been due to the fact that any term added by the compound to the closed time distribution was similar in value to the terms present in control, i.e., the  -222occupation time of L-687,384 in its blocking site is smaller than the normal closed time of the channel in control. Additionally, in the microspectrofluorimetric experiments, the reduction of NMDA-evoked [Ca ]j responses by L-687,384 quickly reached equilibrium 2+  (no use-dependence was observed) and the blocking effect was quick to wash out following superfusion with control solution. A high off-rate constant for L-687,384 is thus suggested, and is consistent with its low potency to reduce P and NMDA-evoked 0  [Ca ]i increases. Correlations between values for off-rate constants and 2+  IC50  values  have been observed previously for a number of NMDA blockers, including P C P , dizocilpine, and ketamine (MacDonald et al., 1991) and a series of memantine derivatives (Parsons et al., 1995). The present results with L-687,384 are therefore consistent with descriptions of open channel blocker properties in other laboratories.  4.3. J. 3.Relationship between open channel block and o~ effects  The NMDA antagonist action of L-687,384 observed in the present study is unlikely to account for the absence of potentiation of NMDA responses reported following 'high' (500 pg kg' i.v.) doses of the drug in vivo (Bergeron et al., 1992). In the 1  case of DTG, a dose of 1 pg kg' i.v. (corresponding to a theoretical maximal brain 1  concentration of < 5 nM; Monnet et al., 1990) potentiates neuronal firing induced by NMDA, whereas 5 mg kg' (corresponding to a maximal concentration in brain of 25 1  pM) fails to do so (Monnet et al., 1992). The IC value for DTG as an antagonist of 50  NMDA-evoked currents under whole-cell voltage clamp is 37 pM (Fletcher et al., 1993), suggesting that functional NMDA receptor antagonism may underlie the failure of high doses of DTG to potentiate the NMDA response in vivo (Monnet et al., 1990; Monnet et  -223a/., 1992). Brain concentrations of L-687,384 following peripheral administration are unknown, but a dose of 500 ug kg" i.v. would theoretically produce a maximal brain 1  concentration of about 1 pM, considerably lower than the apparent  IC50  value for the  reduction of NMDA-evoked rises in [Ca ]i. Unlike DTG, the actions of L-687,384 at the 2+  NMDA channel are evident only at more than 1000-fold higher concentrations than the concentrations required for displacing [ H] 3  ligands from the a recognition site  (Middlemiss etal., 1991; Barnes etal., 1992).  -2245.  SUMMARY This study has incorporated a multi-faceted approach to study the interactions of  four potential neuroprotectant compounds acting at the NMDA receptor-ion channel complex. The compounds chosen belong to classes of agents which encompass ones that are known to possess NMDA antagonist properties, including benzomorphans (the enantiomers (-)- and (+)-p-cyclazocine), and a ligands (DM and L-687,384).  The  approach used is novel in that electrophysiological measurements have been combined with optical recordings to yield biophysical data on mechanisms of action. In turn, the elucidation of mechanisms have aided in the concentration-response  data  regarding  neuroprotectants against excitotoxicity.  the  direct  effects  interpretation of  the  of the  agents  as  Furthermore, ancillary whole-cell recordings  have allowed some insight into possible side effects of the compounds through actions mediated via voltage-gated ion channels. A number of conclusions can be drawn from this work. These are summarized as follows. 1. (-)-p-cyclazocine exhibits marked neuroprotective activity against EAAinduced toxicity on cultured hippocampal and cortical neurons.  The  protection of neurons evinced by (-)-p-cyclazocine is strong evidence that alteration in single-channel behaviour by this agent would result  in  neuroprotection where NMDA overactivity is a key determinant of cellular toxicity. 2. The basis for the agents' neuroprotection is uncompetitive open channel blockade of the NMDA ion channel.  The  IC50  values for open channel  blockade by (-)- and (+)-p-cyclazocine paralleled the  ED50  values obtained in  -225the neuroprotection assays. The E D  50  for open channel block by DM was  higher than that obtained with (-)-p-cyclazocine and accounts for its lower potency in the neuroprotection assays documented by Choi (1987). 3. The magnitudes of the onward (blocking) rate constants are similar for (-)-pcyclazocine, DM, and L-687,384, yet potencies of the compounds for open channel NMDA blockade differ markedly and present in a different rank order than that for k . 2  Thus, the magnitude of the onward rate constant is not a  critical determinant of an agents' potency to block NMDA ion channels. 4. The magnitude of the unblocking rate constant k. for (-)-p-cyclazocine and 2  DM is important in determining the potency of open channel blockade. The k. 2  values for both compounds are much higher than that found for MK-801  (Huettner and Bean, 1988), showing that the former compounds dissociate much faster from their blocking site relative to the latter compound.  This  finding is significant because the very slow offset time of MK-801 is likely a major contributor to the serious neurological side effects produced by this agent. 5. Cellular responses with fura-2-loaded neurons showed use-dependence associated with the onset of (-)-p-cyclazocine action.  This feature of the  antagonist profile indicates that the open NMDA channel is the target, consistent with the patch clamp data.  Use-dependent activity would be  expected to be advantageous for a neuroprotectant, in that the agent would be more efficacious under situations of excessive receptor activation, as in excitotoxic conditions.  -2266. The microspectrofluorimetic studies were also useful in determining the selectivity of the agents for NMDA ion channels. The results indicated (-)-pcyclazocine and DM had no significant actions against non-NMDA (i.e. kainate and AMPA) ion channels. This point is important because such interactions could compromise the clinical utility of the compounds (for example, interference with fast synaptic transmission mediated by kainate and AMPA receptors). In addition, (-)-p-cyclazocine had negligible effects on voltage-gated C a to block C a  2 +  2+  channels. It was noted, however, that DM had an action  channels at concentrations about 4x the IC o for block of NMDA 5  ion channels. 7. Whole-cell patch clamp experiments showed (-)-p-cyclazocine had no significant effects on voltage-gated N a or K* currents in neurons and cardiac +  cells.  The recorded K* currents likely represent ones active during the  repolarization phase of the action potential, including transient and delayed rectifier components. DM had actions to inhibit both Na* and K* currents in cardiac, but not neuronal, cells. This result would suggest the possibility that DM could produce cardiac side effects at concentrations at which it would exert neuroprotective effects. 8. The use of temperature variations is suggested to generally be a useful biophysical probe to study the interactions of agents with sites associated with ion channels. In this work, the relatively low Q  1 0  for the temperature  dependence of NMDA channel conductance would suggest diffusion-limited current flux.  The considerable temperature dependence associated with  -227channel blockade kinetics (Q10 near 3) would suggest utility in both increasing and decreasing temperature to study channel interactions. Unfortunately, the variable temperature studies on kinetic behaviour in the present work were complicated by two findings. Firstly, the kinetic behavour of the NMDA channel, in the absence of channel blockers, was quite strongly dependent on temperature. Secondly, low temperature caused a significant decrease in the frequency of channel events. This effect, when combined with the actions of (-)-(J-cyclazocine and DM to reduce frequency of openings, did not allow for resolution of sufficient events for kinetic distributions. The utility of (-)-p-cyclazocine as a candidate neuroprotectant is strongly indicated by the results of this work.  However, the issue of  drug-induced  psychotomimesis that has been previously encountered with other non-competitive NMDA antagonists must be considered before proceeding with further research into this agent's clinical role in the treatment of excitotoxic brain damage. Use of the openchannel NMDA blocker P C P is associated with unpredictable psychotic symptoms emerging some time following its administration (Willetts et al., 1990). This feature of PCP action is a complication that has led to suspicion that psychotomimesis induced by compounds of this type is a function of NMDA blockade itself, and hence would present an inescapable side effect of NMDA antagonists (Willetts et al., 1990). However, the present study has shown DM, which is not psychotomimetic in humans (Tortella et al., 1989), to exhibit NMDA channel-blocking properties. Furthermore, P C P is not selective for NMDA ion channels; this agent exhibits considerable actions to inhibit voltage-gated  -228Ca  2 +  channels (ffrench-Mullen and Rogawski, 1992), acetylcholine-gated channels  (Aguayo and Albuquerque, 1986), and N a and K* channels (Tourneur et al., 1982). +  These additional actions of P C P may also contribute significantly to the generation of side effects by this compound. As noted above, optical measurements showed (-)-pcyclazocine had no action to block voltage-gated C a cortical neurons.  2+  channels in hippocampal or  In addition, whole-cell patch clamp measurements showed the  compound did not modify N a or K* currents in excitable cells (neurons or cardiac +  myocytes). In conclusion, the rescue of hippocampal neurons from NMDA-induced cell death by low concentrations of (-)-p-cyclazocine suggests further study with respect to its potential as a neuroprotectant compound. The same conclusion is possible with DM with the proviso that the safety margin between neuroprotectant actions and effects on other voltage-gated channels is not large.  The involvement of NMDA receptors in  several neuropathologies, including epilepsy, neurodegeneration following episodes of ischaemia, hypoxia, hypoglycaemia, or C N S trauma, and degenerative disorders such as Huntington's chorea, Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis, has been targetted as a possible focus for therapeutic intervention (Choi, 1988a; Danysz et al., 1995).  The evident uncompetitive nature of (-)-p-  cyclazocine antagonism of NMDA activity in hippocampal neurons suggests that drug efficacy would be enhanced with escalating levels of glutamate. This point is important because drug effects on synaptic transmission mediated by normal levels of glutamate would be minimized, whereas NMDA receptors exposed to relatively high glutamate concentrations would be selectively targetted.  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