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Pre- and postsynaptic actions of pentobarbital on corticothalamic transmission Ran, Israeli 2005

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PRE- A N D POSTSYNAPTIC ACTIONS OF P E N T O B A R B I T A L O N CORTICOTHALAMIC TRANSMISSION by Israeli Ran B . S c , Tel A v i v University, 1995 M . S c . , University o f Calgary, 1998  A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T OF T H E R E Q U I R E M E N T F O R T H E D E G R E E OF D O C T O R OF PHILOSOPHY  in  T H E F A C U L T Y OF G R A D U A T E STUDIES  Neuroscience  T H E U N I V E R S I T Y OF BRITISH C O L U M B I A  July 2005  © Israeli Ran, 2005  Abstract This thesis examined the pre- and postsynaptic actions o f an anesthetic barbiturate, pentobarbital, on neurons o f the corticothalamocortical system in vitro.  The in vivo  system mediates conscious and sleep states. The thesis focuses on pentobarbital actions that induce network oscillations, and modify responses o f single thalamocortical neurons to corticothalamic stimulus trains. The thesis addressed the following: pentobarbital induce oscillations i n thalamic slices? oscillations?  (1) does  (2) what receptors contribute to  (3) how does pentobarbital interact with modulators o f excitability?  (4)  what are pentobarbital effects on post- and presynaptic parameters o f glutamatergic transmission during short-term  depression  (STD)?  (5) how do the  effects  of  pentobarbital on S T D compare with selective action potential blockade? (6) given the well-known actions o f pentobarbital on metabolism, do its effects on S T D mimic glucose deprivation?  Pentobarbital at a subanesthetic concentration induced 1-15 H z oscillations, requiring glutamatergic excitation, but not elevated temperature or low extracellular [ M g ] . 2+  Glycine receptors mediated oscillations i n ventrobasal nuclei, disconnected from nucleus reticularis thalami (nRT). y-aminobutyrate ( G A B A ) receptors mediated oscillations i n isolated n R T .  B y acting on N-methyl-D-aspartate ( N M D A ) receptors, spermine modulated membrane rectification, firing threshold, and decay o f excitatory postsynaptic potentials (EPSPs). These interactions occurred at the polyamine site on N M D A receptors.  Pentobarbital enhanced S T D o f excitatory postsynaptic currents (EPSCs) by decreasing quantal size. These use-dependent effects persisted during blockade o f desensitization and saturation o f glutamate receptors and hence, likely were presynaptic. decreased  Pentobarbital  apparent quantal size and amplitude in the post-stimulus train, evoked  miniature E P S C s (minEPSCs) but not ongoing, pre-train m i n E P S C s , reaffirming a presynaptic action. Pentobarbital eliminated E P S C facilitation early i n a train, due to high extracellular [ K ] ( [ K ] ) . Partial blockade o f action potentials by tetrodotoxin reduced +  +  e  the apparent quantal size and evoked m i n E P S C size, without effect on pre-stimulation m i n E P S C . Like pentobarbital, glucose deprivation reduced quantal size and rundown o f quantal contents.  Glucose deprivation abolished S T D and intra-train, post-gap jump i n  E P S C amplitude.  In summary, this thesis describes several new types o f synaptic modulation by pentobarbital that complement known postsynaptic mechanisms o f anesthesia.  The  analysis techniques provide a new approach for examining the pre- and postsynaptic drug effects on transmission in the brain.  Table of Contents Abstract  —  ii  Table o f Contents  iv  List o f Tables  viii  List o f Figures  x  Abbreviations  xiii  Acknowledgements  xv  Chapter 1. Introduction  1  1.1. Scope o f thesis  1  1.2. Background  2  1.2.1. E E G activity and brain oscillations  2  1.2.2. Receptor mediation o f sleep-like oscillations in the C T C system 1.2.3. Non-synaptic mechanisms o f sleep-like oscillations 1.2.4. Synaptic composition o f the C T C system 1.2.5. G A B A receptors modulate the frequency o f sleep-like oscillations 1.2.6. Glycine receptors contribute to thalamic oscillogenesis 1.2.7. Barbiturate anesthetics induce sleep-like oscillations 1.2.8. Polyamine modulation o f barbiturate action 1.2.9. Polyamine enhancement o f corticothalamic transmission: Relevance to thalamic oscillogenesis 1.2.10. Pre- and postsynaptic mechanisms o f short-term depression (STD) 1.2.10.1. Presynaptic mechanisms in S T D 1.2.10.1.1. Depletion o f quantal store 1.2.10.1.2. Reduction i n transmitter content 1.2.10.1.3. Modification o f the presynaptic action potential 1.2.10.1.4. Inactivation o f C a channels 1.2.10.1.5. Interference with endocytosis 1.2.10.2. Postsynaptic mechanisms o f S T D 1.2.10.2.1. Receptor desensitization 1.2.10.2.2. Receptor saturation 1.2.11. Pre- and postsynaptic effects o f pentobarbital 1.2.11.1. Postsynaptic effects 1.2.11.1.1. Effects on receptor systems 1.2.11.1.2. Effects on non-receptor systems 1.2.11.2. Presynaptic effects o f barbiturates 1.2.11.2.1. Effects on ion channels  3 4 5 6 7 7 8  2 +  - iv -  8 11 11 12 • 15 15 • 15 16 16 16 18 19 19 19 20 20 21  1.2.11.2.1.1. N a channels 1.2.11.2.1.2. Voltage-gated C a channels 1.2.11.2.1.3. K c h a n n e l s 1.2.11.2.2. Effects on the release machinery 1.2.11.2.3. Effects on energy metabolism 1.2.11.2.4. Effects on transmitter release 1.2.11.2.4.1. Evoked release 1.2.11.2.4.2. Spontaneous release +  2 +  +  21 21 21 23 23 • 24 24 25  1.2.12. Theory o f depletion model and fluctuation analysis  26  1.3. Rationale  29  1.4. Objectives and research approach  31  1.5. Major questions  34  Chapter 2. Methods  36  2.1. Slice preparation  36  2.2. Drug application  38  2.3. Extracellular recording  38  2.4. Whole-cell recording  39  2.5. Detection o f signals  40  2.5.1. Direct method 2.5.2. Deconvolution method 2.5.3. First and second derivative method  40 40 41  2.6. Repetitive stimulation  43  2.7. Induction o f plastic modifications o f corticothalamic synaptic responses  43  2.8. Analysis o f extracellular recordings  44  2.9. Fluctuation analysis o f corticothalamic synaptic responses  44  2.10. Statistical comparisons  51  Chapter 3. Results  52  - V -  Part I. Pentobarbital oscillations in vitro in ventrobasal thalamus  52  3.1. Extracellular effects o f pentobarbital 3.1.1. Pentobarbital application and internal capsule stimulation 3.1.2. Effects o f reduced extracellular M g 3.1.3. Effects o f raised temperature 3.1.4. Application o f a high dose o f pentobarbital 3.1.5. Effects o f synaptic receptor blockade 3.1.6. Pentobarbital-induced oscillations i n separated thalamic nuclei 3.1.7. Discussion  52 52 • 52 55 55 58 61 66  Part II. Modulation o f N M D A receptors i n corticothalamic transmission  68  3.2.1. Effects o f spermine • 3.2.1.1. Tonic firing 3.2.1.2. Passive membrane properties 3.2.1.3. Action potential threshold 3.2.1.4. Membrane rectification 3.2.1.5. L o w threshold C a spike (LTS) 3.2.1.6. Excitatory and inhibitory postsynaptic potentials  68 68 69 71 75 80 84  2 +  2 +  firing  3.2.2. Pentobarbital effects on corticothalamic EPSPs  90  3.2.3. Interactions o f Z n  91  3.2.4.  2 +  with spermine and pentobarbital  Antagonism o f polyamine site  93  3.2.5. Discussion  93  Part III. Effects o f pentobarbital on short-term depression  99  3.3.1. Behaviour o f E P S C s in trains during short-term depression ( S T D ) 3.3.1.1. Passive membrane properties 3.3.1.2. Frequency - dependent fade (STD) o f corticothalamic E P S C s  99 99 100  3.3.2. Effects o f alterations in extracellular C a 3.3.2.1. L o w [ C a ] perfusion 3.3.2.2. Elevated [ C a ^ e perfusion  107 107 108  2 +  concentration ([Ca ] ) 2+  e  2+  e  3.3.3. Receptor desensitization and saturation 3.3.3.1. Effects o f blockade o f receptor desensitization 3.3.3.2. Combined blockade o f receptor desensitization and saturation 3.3.4. Effects o f pentobarbital on S T D 3.3.4.1. E P S C behaviour in trains 3.3.4.2. S T D i n raised Ca concentration 2+  - vi -  • 113 113 113 117 117 122  <  3.3.4.3. S T D i n reduced Ca concentration 3.3.4.4. Combined cyclothiazide and kynurenate blockade 3.3.5. Effects o f altered extracellular K concentration ( [ K ] ) 3.3.5.1. H i g h [ K ] perfusion 3.3.5.2. L o w [ K ] perfusion 2+  +  +  e  +  e  +  e  127 • 128 128 132 133  3.3.6. Effects o f tetrodotoxin  139  3.3.7. Effects o f glucose deprivation on S T D  148  3.3.8. Discussion.  154  4. General discussion  163  Bibliography  182  - vii -  List of Tables 1.1. Summary o f synaptic and non-synaptic actions o f pentobarbital  22  2.1. Comparison of peak detection methods  42  3.1. Effects o f spermine on E P S P variables  86  3.2.1 A . Summary of parameters o f corticothalamic S T D at different frequencies  102  3.2. I B . Derived parameters o f S T D at different frequencies  106  3.2.2A. Summary o f effects o f altered [ C a ] on parameters o f S T D  Ill  3.2.2B. Effects o f altered [ C a ] on derived parameters o f S T D  112  3.2.3 A . Summary o f effects o f C T Z and K Y N on parameters o f S T D  115  3.2.3B. Effects o f C T Z and K Y N on derived parameters o f S T D  116  3.2.4A. Summary of pentobarbital effects on parameters o f S T D  119  3.2.4B. Effect of pentobarbital on derived parameters o f S T D  121  2+  e  2+  e  3.2.5A. Pentobarbital effects on parameters o f S T D i n raised [ C a ]  123  2+  e  3.2.5B. Pentobarbital effects on derived parameters o f S T D in raised [ C a ] 2+  3.2.6A. Pentobarbital effects on parameters o f S T D i n low [ C a ]  e  124 125  2+  e  3.2.6B. Pentobarbital effects on derived parameters o f S T D i n low [ C a ] 2+  e  126  3.2.7A. Pentobarbital effects on parameters o f S T D during co-applied C T Z and K Y N 130 3.2.7B. Pentobarbital effects on derived parameters o f S T D during co-applied C T Z and KYN  131  3.2.8A. Summary o f effects of high [ K ] , pentobarbital on parameters o f S T D  135  3.2.8B. Derived parameters o f S T D for high [ K ] , pentobarbital  136  3.2.9A. Summary o f effects o f low [ K ] , pentobarbital on parameters o f S T D  137  +  e  +  e  +  e  - viii -  3.2.9B. Derived parameters o f S T D i n low [ K ] , pentobarbital  138  3.2.1 OA. Summary o f T T X effects on parameters o f S T D  143  3.2.10B. T T X effects on derived parameters o f S T D  144  3.2.11 A . T T X effects on parameters o f S T D during co-applied C T Z + K Y N  146  +  e  3.2.1 I B . T T X effects on derived parameters o f S T D during co-applied C T Z and K Y N 147 3.2.12 A . Summary o f parameters o f S T D at different glucose concentrations  • 151  3.2.12B. Derived parameters o f S T D at different glucose concentrations •  153  4.1. Receptor involvement in pentobarbital-induced oscillations  167  List of Figures 1.1. Diagram o f the corticothalamocortical circuit  5  1.2. Depression o f end-plate potentials (EPPs) during tetanic stimulation  13  1.3. Intra- and intersite variability o f transmitter release  14  1.4. Mechanisms o f short-term depression and their presumed site o f action  17  2.1. Direct method o f peak detection  40  2.2. Peak detection obtained by using deconvolution method  41  2.3. First and second derivative method for peak detection  41  3.1.  Pentobarbital  induces  extracellular oscillations in ventrobasal  electrical stimulation o f internal capsule at 0.05 H z 3.2. Pentobarbital-induced oscillations i n low M g  2 +  53  medium  54  3.3. Effects o f raised temperature on pentobarbital oscillations 3.4.  Time dependence o f effects oscillations  o f increasing concentrations  nuclei during  56 on  pentobarbital 57  3.5. Antagonists o f G A B A , and glycine receptors modulate frequency o f pentobarbital oscillations  60  3.6. Photomicrograph o f sagittal slice shows complete separation (asterisk) o f V B nuclei from n R T  63  3.7. Pentobarbital oscillations i n electrically stimulated V B nuclei, after surgical separation from n R T  64  3.8. Pentobarbital oscillations in electrically stimulated n R T , before and after its surgical separation from V B nuclei  65  3.9. Spermine enhanced tonic firing in a concentration-dependent  manner i n M G B  neurons  70  3.10. Spermine (100 u M ) decreased the action potential threshold  72  3.11. Spermine increased tonic firing by interacting with N M D A receptors  74  3.12. Effects o f spermine (100 u M , 3 min) on membrane rectification  77  2+  3.13. Alterations i n extra- and intracellular C a  influence  spermine  effects  depolarizing current - voltage ( V -1) relationships i n M G B neurons 2+  3.14. Effects o f spermine (100 u M , 3 min) on the low threshold C a  spike firing  on 79 83  3.15. Spermine (100 u M , 3 min) prolonged late component o f corticothalamic E P S P s mediated by N M D A receptors  85  3.16. Spermine (100 u M ) prolonged the EPSPs by interacting with the polyaminesensitive site on N M D A receptor  89  3.17. Pentobarbital effects on N M D A - m e d i a t e d corticothalamic EPSPs  92  3.18. Pentobarbital reversal o f spermine E P S P prolongation involves interactions at the polyamine site on N M D A receptor  94  3.19. Frequency-dependence o f corticothalamic S T D  101  3.20. Validation o f the corrected variance-mean method during corticothalamic S T D - 1 0 4 3.21. Pre- and post-stimulation miniature E P S C s vary i n size  105  3.22. Persistence o f S T D i n media - containing low C a  109  3.23. C a  2 +  2 +  modification o f corticothalamic S T D  110  3.24. Effects of blockade o f receptor desensitization and saturation on S T D  114  3.25. Dose-dependence o f pentobarbital enhancement o f corticothalamic S T D  118  3.26. Quantal alterations mediate pentobarbital effects on S T D  120  3.27. Pentobarbital enhancement  o f S T D during combined blockade o f receptor  desensitization and saturation  129  3.28. Effects o f altered K concentration on pentobarbital enhancement o f S T D  134  3.29. Tetrodotoxin enhanced S T D by reducing quantal size  141  +  3.30. T T X decreased the size o f evoked miniatures E P S C s without affecting spontaneous miniature E P S C size  142  3.31. T T X effects on S T D during blockade o f receptor desensitization and saturation .145 3.32. Effects o f glucose deprivation on S T D  150  3.33. Evoked and spontaneous miniature E P S C s during glucose deprivation  152  4.1. Possible synaptic targets for pentobarbital actions during corticothalamic S T D . . . . 181  - Xll -  Abbreviations ACSF  Artificial cerebrospinal fluid  AMPA  a-amino-3-hydroxy-5methyl-4-isoxazoleproprionate  ANOVA APV  Analysis o f variance 2-amino-5-phosphono-valerate  ATP  Adenosine-5'-triphosphate  cAMP  Cyclic 3' 5' -adenosine-monophosphate  CSF  Cerebrospinal fluid  CNQX  6-cyano-7-nitroquinoxaline  CNS  Central nervous system  EC50  Concentration o f drug that produces a half-maximal effect  EEG  Electroencephalogram  EGTA  Ethylene glycol-bis-(P-aminoethyl ether) N,N,N N'-tetraacetic acid  EPSP  Excitatory postsynaptic potential  EPSC  Excitatory postsynaptic current  GABA  Y-aminobutyric acid  GluR  Glutamate receptor  h  hour  HEPES  N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]  Hz  Hertz (s" )  IC50  Concentration o f a drug that produces a half-maximal inhibition  I  Hyperpolarization activated inward current  h  Iicir  r  1  Inwardly rectifying K  +  current  - xiii -  Iieak  I  N a P  I  x  Voltage-independent leak current Persistent N a  +  current  L o w threshold C a  2 +  current  IPSP  Inhibitory postsynaptic potential  IPSC  Inhibitory postsynaptic current  LTS  L o w threshold C a  MGB  Medial geniculate body  2 +  spike  min  Minute  NMDA  N-methyl-D-aspartate  nRT  Nucleus reticularis thalami  pH  Hydrogen concentration; pH-log[H ]  pKa  Dissociation constant; pH-log[base]/[cation]  Ri  Input resistance  REM  Rapid eye movement  SEM  Standard error about the mean  T  Time constant; time required to reach (1-1/e) o f a steady state value  TEA  Tetraethylammonium  TTX  Tetrodotoxin  V  Membrane potential  m  +  VB  Ventrobasal complex o f the thalamus  VPL  Ventral posterior lateral thalamic nucleus  - xiv -  Acknowledgements I would like to express m y deepest gratitude to my supervisor Dr. Ernie Puil who not only provided me sheer intellectual and moral support but also encouraged me to pursue m y own original ideas. I am extremely grateful to Dr. David Mathers for the  fruitful  discussions that contributed to the work.  I am extremely and equally grateful to Dr. David Quastel who provided excellent guidance i n analysis and interpretation o f the results. Without his help this work would not have been possible. I wish to thank Dr. T i m Murphy for excellent comments and suggestions.  The present work was supported by the Canadian Institutes for Health Research, Mathematics i n Information Technology and Complex Systems, and the Jean Templeton H u g i l l Foundation.  I thank M s . Viktoriya Dobrovinska for preparation o f materials and solutions and M r . Christian Caritey for excellent technical support. I thank Douglas B r o w n for his assistance with photography o f brain slices.  M y gratitude for invaluable intellectual and emotional support goes to m y family and friends. X i a n g W a n and Amer Ghavanini each deserve a special thank you for sharing a set-up and discussing scientific issues. The assistance o f Mitrut Isbasescu was extremely helpful i n programming and running various software. I'd also like to thank the new  XV  recruits o f our laboratory Sarah McCarthy and Stephanie Lee for asking interesting science-related questions. Last but not least, I'd like to thank m y beloved girlfriend Andrea Krawzcyk whose moral support was needed at many exhausting moments.  - xvi -  Chapter 1. Introduction I Ran  Chapter 1 INTRODUCTION  1.1. Scope o f thesis The thesis describes in vitro experiments that delineate the modulation o f corticothalamic transmission b y pre- and postsynaptic Introduced i n the first half o f the 2 0  th  actions  o f the barbiturate,  pentobarbital.  century, barbiturates have received extensive  clinical use, based on their pharmacological and pharmacokinetic properties. this class are sedative-hypnotics, anti-epileptics, and general anesthetics.  Drugs i n  Occasionally  used i n humans, pentobarbital is still the most widely used general anesthetic i n experimental animals. Pentobarbital produces a wide range o f in vivo effects.  Unlike  other barbiturates, pentobarbital does not have anti-epileptic properties at subanesthetic doses. L i k e other barbiturates i n anesthetic doses, pentobarbital is capable o f terminating convulsions. The anesthetic effects o f pentobarbital are due to a depression o f neuronal excitability i n the central nervous system (CNS). This reduced responsiveness occurs i n all C N S regions, contributing to the overall loss o f awareness o f environment during induction o f pentobarbital anesthesia.  It is generally accepted that barbiturate-induced  depression involves postsynaptic  interactions o f neurons i n the cortico-thalamocortical ( C T C ) system. A s with other drugs, there is limited evidence for presynaptic actions o f pentobarbital at central synapses. This gap i n knowledge stems from the inability to obtain reliable electrical recording at axon terminals, which regulate neurotransmitter release. This thesis explores the method  Chapter 1. Introduction I Ran  -2-  o f fluctuation analysis o f synaptic responses to repetitive stimulation. The methodology described here facilitates the separation o f pre- from postsynaptic drug effects. It provides a new approach for assessing actions o f drugs, such as pentobarbital. The overall hypothesis o f this thesis is that pentobarbital has actions at pre- and postsynaptic sites on neurons; reduction o f excitatory transmitter release compromises transmission i n the C T C system. If these effects occur at anesthetic doses in vivo, they could contribute to a loss o f consciousness.  1.2. B a c k g r o u n d 1.2.1. EEG activity and brain oscillations In the conscious brain, sleep and wake states correspond to varying degrees o f synchronized oscillations in the electroencephalogram ( E E G ) , correlative with rhythmic electrical activity o f networks o f neurons i n the C T C system. Voltage oscillations o f neurons i n the C T C network produce this synchrony (Steriade, 2003). During the early stages o f sleep, there is prevalent spindling E E G activity, which reflects 6-14 H z oscillations o f cortical neurons. A s sleep evolves into deeper stages, this spindling behaviour transforms to the slower delta activity i n the 1 - 4 H z range. Lesions i n the thalamus result i n a disruption o f the characteristic rhythmic E E G pattern i n cortical neurons (Villablanca and Salinas-Zeballos, 1972). Thalamic neurons can generate and maintain oscillations that are independent o f cortical inputs (Villablanca and Marcus, 1972). In summary, the thalamus is an essential component o f the network for generating oscillations during natural sleep.  -3-  Chapter 1. Introduction I Ran  1.2.2. Receptor mediation of sleep-like oscillations in the CTC system The synaptic interactions that mediate thalamic oscillations are complex (Jones, 2002). The type o f receptors involved in generation o f oscillations may alter their frequency range.  For example, antagonism o f ionotropic receptors for a-amino-3-hydroxy-5-  methyl-4-isoxazolepropionic acid ( A M P A ) enhances the light stage o f non-rapid eye movement ( N R E M ) slow wave sleep resulting i n 6-14 H z oscillations. O n the other hand, N-methyl-D-aspartate ( N M D A ) receptor antagonism increases the deep stages while reducing the lighter stage o f N R E M sleep, favoring oscillations i n the 1-4 H z range (Juhasz et al., 1990). Activation o f thalamic metabotropic glutamate receptors results i n 1-4 H z membrane oscillations (Emri et al., 2003). Thus, the involvement o f multiple excitatory receptors i n thalamic oscillogenesis provides a mean for regulating the transition between the light and deep stages o f sleep.  Reduction i n synaptic transmission through ionotropic receptors for the inhibitory transmitter, y-aminobutyric acid ( G A B A ) results in sustained thalamic oscillations. Blockade o f type A G A B A ( G A B A ) receptors with the potent antagonist, bicuculline, A  produces delta (1-4 H z ) oscillations, characteristic o f deep sleep (von Krosigk et al., 1993). Penicillin, a much weaker G A B A  A  receptor antagonist, is also effective i n  inducing sleep oscillations in vivo (Vital-Durand et al., 1972). In both cases, the oscillations result, presumably, from a reduced inhibitory receptor-mediated transmission, which heightens neuronal excitability and primes the C T C system for rhythmic activity.  Chapter 1. Introduction I Ran  -4-  1.2.3. Non-synaptic mechanisms of sleep-like oscillations Non-synaptic interactions  provide  an  additional mechanism  to  explain  thalamic  oscillations. These mechanisms include intrinsic ionic conductances and gap junctions. According to this view, oscillations initiate with a depolarization o f thalamic neurons which brings their resting membrane potential above threshold and generates regenerative Ca  2 +  spikes. Upon inactivation o f the C a  2 +  channels, activation o f voltage sensitive K  +  channels hyperpolarizes the membrane potential, which is then repolarized by the hyperpolarization-activated  cation  conductance  (IR).  The  contribution  of  ionic  conductances has been demonstrated in vitro and was obtained by the use o f patch clamp recordings i n thalamic neurons (Leresche et al., 1991, M c C o r m i c k and Pape, 1990). In these studies, oscillations occurred i n isolated neurons that express T-type and L-type 2+  Ca  channels and were independent o f synaptic inputs (Alonso and Llinas, 1989;  Leresche et al., 1991). Pharmacological blockade o f these channels abolished the ability to generate rhythmic patterns.  M o r e recent studies (Hughes et al., 2002) have implicated gap junctions as mediators o f thalamic synchrony. This appears to be the case i n a small subset o f ventrobasal thalamic neurons, but may not be significant for thalamic synchronization. Hence, intrinsic conductances are an essential requirement for generation o f synchronized activity i n thalamic neurons i n addition to synaptic components o f the C T C system.  Chapter 1. Introduction I Ran  -5-  1.2.4. Synaptic composition of the CTC system The C T C system consists o f excitatory glutamatergic and inhibitory G A B A e r g i c pathways, as well as 'presumed' glycinergic pathways (Ran et al., 2004). Thalamocortical relay neurons, i n the dorsal thalamic nuclei, receive extensive connections from the  9lu?  Thalamocortical neuron  Intemeuron  Dorsal thalamus  j;  /  Figure 1.1. Diagram o f the corticothalamocortical circuit. Thalamocortical relay neurons i n the dorsal thalamus receive excitatory glutamatergic inputs from pyramidal neurons i n the neocortex. In the neocortex, pyramidal neurons receive reciprocal excitatory glutamatergic inputs from thalamocortical relay neurons. G A B A e r g i c neurons i n the reticular thalamic nucleus receive glutamatergic inputs from pyramidal neurons i n the neocortex and from thalamocortical relay neurons. Reticular neurons send inhibitory projections to thalamocortical relay neurons i n the dorsal thalamus. G A B A e r g i c reticular neurons contain local inhibitory collateral and dendro-dendritic synapses.  Chapter 1. Introduction I Ran  -6-  cortex v i a glutamatergic synapses which constitute the corticothalamic pathway (Figure 1.1). The projection o f thalamocortical neurons back to the cortex is a reciprocal pathway, mediated by glutamate (Jones, 2002). There is also a unidirectional excitatory glutamatergic pathway which originates i n the cortex and terminates i n the reticular thalamic nucleus. In addition, neurons  o f the dorsal thalamus  excite inhibitory  G A B A e r g i c reticular thalamic neurons by a glutamatergic pathway (Ohara, 1988). Thus, corticothalamic transmission triggers a loop o f excitatory and inhibitory inputs i n the C T C system.  Corticothalamic activation occurs at ionotropic and metabotropic glutamate receptors (Jones, 2002). Ionotropic receptors include A M P A / k a i n a t e and N M D A receptors that mediate fast and slow excitatory postsynaptic responses, respectively. Slow excitation is also possible by activation o f metabotropic glutamate (mGlu) receptors. Overall, a wide spectrum o f excitatory receptors that are subject to modulation, together with inhibitory receptors, govern the pattern o f C T C rhythmic activity.  1.2,5. GAB A receptors modulate the frequency ofsleep-like oscillations Inhibitory transmission, mediated by ionotropic G A B A  A  receptors and metabotropic  GABA  B  receptors, originates from G A B A neurons o f the nucleus reticularis thalami  (nRT)  which terminate on dorsal thalamic neurons (reviewed by Llinas et al., 2005).  There are inhibitory interneurons intrinsic to the dorsal thalamus that are estimated to make up 1-2 % o f the total neuronal population. Activation o f G A B A A and G A B A B receptors contributes to the transition between the delta and spindle rhythms (Bal et al.,  Chapter 1. Introduction I Ran  -7-  1995a,b). G A B A A receptors have been implicated i n the generation o f spindle rhythms (von Krosigk et al., 1993). G A B A  B  receptors are thought to exist extra-synaptically and  mediate the slower delta (1-4 H z ) oscillations (Bal et al., 1995a). The different durations and locations o f G A B A  A  and G A B A B receptor mediated responses provide frequency  modulation.  1.2.6. Glycine receptors contribute to thalamic oscillogenesis A few studies have implicated glycine receptors i n thalamic inhibition i n addition to G A B A receptor mediated inhibition (Tebecis, 1974; Ghavanini et al., 2005). Glycine receptors have been shown to mediate theta-like oscillations (6-15 H z ) induced b y pentobarbital i n isolated thalamic ventrobasal slices (Ran et al., 2004). This novel pathway is quite intriguing, in light o f the fact that glycine receptors mediate oscillations independent o f G A B A transmission i n spinal neurons.  Glycine receptor antagonism  induces oscillations i n other systems, such as i n spinal cord neurons (Bracci et al., 1996). Thus, the rhythmic activity o f the C T C network may be shaped by at least two types o f inhibitory ionotropic receptors.  1.2.7. Barbiturate anesthetics induce sleep-like oscillations Barbiturates induce cortical as well as thalamic oscillations, in vivo, similar to those during natural sleep. This ability is attributable to changes i n membrane properties o f neurons i n the C T C system. The oscillations occur at frequencies i n the delta (1-4 H z ) and theta (7-14 H z ) ranges, similar to deep and/or light natural sleep. The most commonly used barbiturates with this property include pentobarbital and thiopental. Less  Chapter 1. Introduction I Ran  -8-  commonly, volatile anesthetics induce in vivo oscillations (Keifer et al., 1994). One o f the mechanism by which barbiturates induce oscillations may depend on the excitable state of  thalamic neurons,  involving modulation o f membrane  properties  and  receptor  interactions at corticothalamic synapses.  1.2.8. Polyamine modulation of barbiturate action The  anesthetic  potency  o f barbiturates  transmission through N-methyl-D-aspartate  depends  on  actions  on  corticothalamic  ( N M D A ) receptors, subject to polyamine  modulation. For example, polyamines decrease the rate at which N M D A receptors desensitize (Lerma, 1991) whereas pentobarbital has the opposite effect (Charlesworth et al.,  1995). However, the effects  o f polyamines at N M D A  receptors include both  enhancement and inhibition (Benveniste and Mayer, 1993). Spermine, among other polyamines, enhances the property o f barbiturates to induce general anesthesia. This enhancement may result from dual inhibitory actions o f spermine and pentobarbital at the Mg  2 +  site on N M D A receptors (Daniell, 1992). However, pentobarbital depression o f  corticothalamic transmission may prevent the prolongation o f N M D A responses caused by spermine.  1.2.9. Polyamine enhancement of corticothalamic transmission: Relevance to thalamic oscillogenesis Polyamines are endogenous biogenic amines that interact with synaptic and nonsynaptic targets and may modulate corticothalamic transmission or enhance thalamic excitability. The C T C circuit may be primed for oscillatory activity as a result o f polyamine-mediated  Chapter 1. Introduction I Ran  -9-  prolongation o f EPSPs. Similar to other polyamines, spermine acts at both sides o f neuronal membrane. These interactions influence ion channels and channels (reviewed by Williams, 1997).  transmitter-gated  Intracellular spermine enhances membrane  rectification by blocking inward rectifier K (KFR) channels (Schuber, 1989) and confers +  an inwardly rectifying property on receptor-gated channels activated by acetylcholine (Haghighi and Cooper, 2000) and A M P A ( K o h et al., 1995). Extracellularly, spermine interacts  with receptors for N M D A  resulting in the prolongation o f postsynaptic  responses (reviewed by Rock and Macdonald, 1995). L o w concentrations o f spermine enhance N M D A - e v o k e d  currents  whereas high concentrations  produce  a voltage-  dependent block o f these currents i n hippocampal neurons (Benveniste and Mayer, 1993). The effects o f spermine and its ability to influence barbiturate actions are unknown i n the C T C system.  Spermine and other polyamines are present at micromolar extracellular concentrations i n the brain, including the thalamus (Harman and Shaw, 1981).  This may indicate a  possible role as a modulator o f membrane excitability. Recent binding studies have challenged the validity o f the previous measurements and estimate the extracellular concentration o f spermine at < 1 u M (Dot et al., 2000). spermine  Neurons and glia release  during electrical stimulation, depolarization by high external  [ K ] , and +  activation o f NMDA-receptors (Harman and Shaw, 1981; Fage et al., 1992). Uptake o f spermine maintains low extracellular concentrations,  presumably resulting from a  constitutive release o f spermine (Dot et al., 2000). Spermine uptake is regulated by various transporters in the dendrites, cell bodies, and nerve terminals, as well as by a high  Chapter 1. Introduction I Ran  - 10 -  affinity transporter expressed i n glia (Laube and V e h , 1997). Spermine, at concentrations > 50 u M , enhances long-term potentiation (Pussinen et al., 1998; Toth et al., 2000) and neuroprotection (Trout et al., 1993; M u i r and Lees, 1995; Ferchmin et al., 2000). Hence, alterations i n extracellular spermine concentration can provide a unique mechanism for modulation,  prolonging  or  shortening  the  duration  of  excitatory  responses  to  corticothalamic stimulation.  A deficiency o f spermine may have important consequences in dysfunctional states, whereas excessively high extracellular concentrations may predispose neurons i n the C T C network to hyperexcitable states.  For example, pharmacological inhibition o f  polyamine synthesis decreases polyamine concentrations in the cochlea (Schweitzer et al., 1986).  This deficiency induces a temporary hearing loss i n humans and rats  (reviewed by M c C a n n and Pegg, 1992), possibly due to decreased spermine-regulation o f NMDA-receptor-mediated activities i n cochlear neurons (Petralia et al., 2000). other hand, exceptionally high concentrations  O n the  o f spermine may exist i n several  neurological disorders, including neurodegenerative diseases (Yatin et al., 2001), stroke (Lukkarinen et al., 1997), global and focal ischemia (Baskaya et al., 1997; Dogan et al., 1999) and kindling epilepsy i n an animal model (Hayashi et al., 1992; Herberg et al., 1992; De Sarro et al., 1993; Halonen et al., 1993). Hence, the alterations o f excitable states imposed by endogenous modulators may determine the outcome o f barbiturate actions  on thalamic neurons.  However, barbiturates may modify corticothalamic  transmission independent o f the excitable state o f thalamic neurons during short-term alterations o f synaptic strength or, short-term plasticity.  Chapter 1. Introduction I Ran  -11-  1.2.10. Pre- and postsynaptic mechanisms of short-term depression (STD) Synaptic plasticity has long been the focus o f many neuroscientists, i n view o f its potential roles i n signal processing, learning, and memory (Fortune and Rose, 2000; Zucker  and Regehr, 2002).  enhancement  and  reduction  Plastic changes in synaptic efficacy include both o f neurotransmission,  depending  upon  experimental  conditions and stimulation paradigms. Enhancement can be observed both on short- and long-time scales, termed facilitation and long-term potentiation (LTP), respectively, and reduction also can be short- (seconds to minutes) or long-lasting (minutes to hours), termed short- and long-term depression ( S T D and L T D ) , respectively (von Gersdorff and Borst, 2002; Voronin, 1994). Both facilitation and depression are demonstrable on short time scales i n synaptic responses evoked by pairs o f evoked stimuli, generally referred to as paired-pulse facilitation or paired-pulse depression.  These plastic alterations also  occur during and after trains o f stimuli at certain frequencies o f stimulation. Although paired-pulse studies have shed light on short time scale modifications o f synaptic strength, the dynamics and progression o f these phenomena are rather limited.  Studies  that use intermediate (5-20 pulses) to long (>20 pulses) trains o f stimulation pulses have provided a more comprehensive view o f short-term depression and a fuller expression o f these processes (reviewed by von Gersdorff and Borst, 2002).  1.2.10.1. Presynaptic mechanisms of STD S T D reflects reductions in the number o f quanta released per stimulus (Elmqvist and Quastel, 1965a). This could be because o f depletion o f a releasable presynaptic store o f quanta, consequent to release, as proposed by Liley and North (1953). However, there are  Chapter 1. Introduction I Ran  other possibilities: inactivation o f presynaptic C a  -12-  2 +  channels, changes i n presynaptic  action potential configuration, or decreased rate o f endocytosis (see below). A reduction i n the amount o f transmitter per quantum would also be classified as presynaptic (Elmqvist and Quastel, 1965b).  It should be noted that it is often assumed that there is a 1:1 relationship between quantum and vesicle (del Castillo and Katz, 1954). This relationship has been confirmed by increases i n membrane capacitance measured during vesicular fusion (Aimers and Neher, 1987). However, the relationship is i n fact controversial (Matthews, 1996; Vautrin and Barker, 2003). For the present purposes, a quantum is defined as an elementary pulse o f a packet o f transmitter which generates a brief synchronized postsynaptic current.  Presynaptic mechanisms contribute to, or dominate, S T D at low to moderate stimulus frequencies (< 10Hz ; Elmqvist and Quastel 1965a,b; Charlton et al., 1982; Emptage et al., 2001). However, at higher stimulation frequencies, postsynaptic mechanisms such as receptor desensitization or saturation, may also contribute to S T D (Figure 1.4).  1.2.10.1.1. Depletion of quantal store A simple model that assumes the release o f a constant fraction o f 'available quanta' with each action potential, at a constant rate o f 'refill', might explain S T D on the basis o f store depletion (Vere-Jones, 1966). These assumptions are too simplistic for neuromuscular (Elmqvist and Quastel, 1965a) or hippocampal synapses (Rosemund and Stevens, 1996) and the calyx o f Held ( W u and Borst, 1999; Schneggenburger et al., 1999). For example,  Chapter 1. Introduction I Ran  - 13-  the rate o f refill o f transmitter packets is evidently accelerated at high frequencies o f stimulation (Elmqvist and Quastel, 1965a; Wang and Kaczmarek, 1998), resulting i n a faster recovery from depletion o f the readily releasable pool o f transmitter packets. The adjustment o f the rate o f refill o f transmitter packet may provide a regulatory mechanism that diminishes S T D i n response to high frequency inputs.  Figure 1.2: Depression o f end-plate potentials (EPPs) during tetanic stimulation. Extracellularly recorded EPPs decrease and reach a plateau after 8 stimuli. Plateau reflects a steady-state between depletion o f transmitter packets and refill o f quanta and o f transmitter per quanta. Stimulation frequency was 180 H z (From Liley and North 1953).  The release probability, presumably the fraction o f 'available quanta' that are released per stimulus, varies in different preparations. Release probabilities have a heterogeneous distribution at different release sites ( W u and Borst, 1999; Sakaba and Neher, 2001). The heterogeneity o f release probabilities could depend on the position o f transmitter packets with respect to C a  2 +  channels (positional heterogeneity) and/or their sensitivity to C a  (biochemical heterogeneity).  2 +  These observations led to the following modifications in  the depletion model (Liley and North, 1953; Elmqvist and Quastel, 1965a, M i l e d i and  -14-  Chapter 1. Introduction I Ran  Thies, 1967): 1) a C a - and activity-dependent enhancement o f refill (Elmqvist and Quastel, 1965a; Zimmermann and Whittaker, 1977); 2) the existence o f multiple groups o f transmitter packets with varying probabilities o f release (Auger and Marty, 1997); and, 3) a decrease i n the number o f participating release sites (Weis et al., 1999).  A n increase i n presynaptic intracellular C a  2 +  concentration ([Ca ];) accelerates the 2+  uptake o f transmitter into packets or increases the number o f releasable packets to the same extent i n the absence or presence o f an exogenous C a 1999). This implies that the actions o f internal C a messengers.  2 +  2 +  buffer ( W u and Borst,  are indirect and may involve second  Indeed, interactions o f [Ca ]j with calmodulin activate protein kinases 2+  which accelerate vesicular refill and reduce store depletion (Sakaba and Neher, 2001).  The existence o f groups o f packets with varying release probabilities does not alter the rate o f store depletion at any one site. Instead, there is a heterogeneous distribution o f groups o f transmitter packets in different release sites (Figure 1.2; Auger and Marty, 1997). Intrasite quantal variance  Intersite quantal variance  Figure 1.3: Intra- and intersite variability o f transmitter release.  Chapter 1. Introduction I Ran  -15-  1.2.10.1.2. Reduction in transmitter content A second possible mechanism for S T D is reduction in amount o f transmitter per quantum (Elmqvist et al., 1965b; Naves and V a n Der Kloot, 2001; reviewed by Vautrin and Barker, 2003). Blocking acetylcholine synthesis reduces the transmitter content early (Van der Kloot and M o l g o , 1994) or only late (Elmqvist et al., 1965b) after prolonged neuromuscular stimulation. A t C N S synapses, there are both supporting (Ishikawa et al., 2002) and contradicting (Sakaba and Neher, 2001) reports about the correlation between reduction i n transmitter content and depression.  Studies on rates o f transmitter refill,  recycling and endocytosis would clarify these discrepancies.  1.2.10.1.3. Modification of the presynaptic action potential A third presynaptic mechanism o f S T D is modification o f presynaptic action potential configuration (Brigant and Mallart, 1983; Smith, 1983). A l s o , i n cultured hippocampal neurons, N a channel inactivation produces failure i n nerve conduction at presynaptic +  branch points, enhancing depression (Debanne et al., 1997; Brody and Y u e , 2000; He et al., 2002). However, these, changes occur i n conjunction with changes i n C a  2 +  currents.  A t the calyx o f Held, action potentials are reduced i n amplitude and increased i n duration much to the same degree as C a  2 +  currents, leading to S T D (Borst and Sakmann, 1999).  Hence, this mechanism may co-exist with Ca -dependent aspects o f S T D . 2+  1.2.10.1.4. Inactivation of Ca channels 2+  A fourth mechanism o f S T D involves enhanced inactivation o f C a  2 +  channels at synaptic  terminals. A t calyx o f Held neurons, the inactivation o f release sites contributes to S T D  Chapter 1. Introduction I Ran  -16-  during prolonged high frequency stimulation (Forsythe et al., 1998). The inactivation o f 9+  Ca  channels is subject to modulation and coupling to various G-proteins by glutamate-  (mGluRs),  GABAB-,  adenosine-,  and  noradrenaline-receptors  (Barnes-Davis  and  Forsythe, 1995; Isaacson, 1998; Kajiwara, 1997; Takahashi et al., 1996; W u et al., 1998). In calyx o f Held neurons, modulation by m G l u R s seems to contribute to up to 10 % o f 9+  depression due to inhibition o f C a inactivation o f C a  2 +  currents (von Gersdorff et al., 1997). In summary,  channels might impair the release process enhancing S T D at  corticothalamic synapses.  1.2.10.1.5. Interference with endocytosis The fifth mechanism, and last to be discussed here, is the regulation o f endocytosis, discovered i n shibire mutant flies. These mutants suffer from severe S T D and paralysis due to defective endocytosis at neuromuscular synapses (Poodry and Edgar, 1979). Recent investigations show an enhanced S T D i n response to genetic interference with endocytosis (Delgado et al., 2000; Luthi et al., 2001). Blockade o f the action o f dynamin, a regulator o f vesicle endocytosis, markedly enhances S T D and prolongs the recovery time at calyx o f Held synapses (Takahashi et al., 2000).  1.2.10.2. Postsynaptic mechanisms of STD 1.2.10.2.1. Receptor desensitization A t high stimulation frequencies, postsynaptic receptor desensitization and saturation contribute to S T D . Receptor desensitization o f A M P A receptors was observed during paired-pulse depression at retinogeniculate synapses (Chen et al., 2002; Kielland and  Chapter 1. Introduction I Ran  17-  Altered Ca"- secretion coupling: - Depletion of releasable vesicles - Inactivation of release sites - Change in sensitivity to C a " - Modulation of vesicle recruitment  Inhibitory autoreceptors: - Metabotropic - Adenosine - Noradrenaline -GABA Reduction of postsynaptic sensitivity: - Desensitization - Saturation  - Lowered excitability  Figure 1.4: Mechanisms o f short-term depression and their presumed site o f action. Different mechanisms o f synaptic depression, acting at distinct points i n the synaptic vesicle cycle have been proposed and can be directly studied i n synapses with large synaptic terminals (Adapted from von Gersdorff and Borst, 2002).  Chapter 1. Introduction I Ran  - 18 -  Heggelund, 2002). However, the long recovery time (>4 s) o f postsynaptic responses after S T D induced by 5 or 10 H z stimulus trains implies that receptor desensitization does not contribute to S T D at all stimulation frequencies.  The faster time scale o f recovery  from desensitization o f A M P A and N M D A receptors means that these receptors do not likely contribute to S T D at low frequencies o f stimulation. Since A M P A and N M D A receptors desensitize on different time scales, a postsynaptic mechanism also would depend on their recovery times. However, the recovery time from S T D is identical i n the responses  evoked on  activation o f both receptors.  This means  that  receptor  desensitization makes minimal contribution to S T D at low frequencies o f stimulation.  1.2.10.2.2. Receptor saturation A saturation o f postsynaptic A M P A receptors might also contribute to S T D . Although a single quantum does not saturate A M P A receptors (Ishikawa et al. 2002), intensive stimulation o f glutamate release leads to significant receptor saturation at calyceal synapses ( W u and Borst, 1999). Non-stationary fluctuation analysis methods reveal a contribution o f receptor saturation to S T D (Scheuss et al., 2002).  L i k e receptor  desensitization, saturation is not significant at low stimulation frequencies and is minimal at the onset o f repetitive stimulation when release o f transmitter is maximal (Matveev and Wang, 2000). Hence, receptor saturation does not likely contribute to depression at l o w stimulation frequencies.  Chapter 1. Introduction I Ran  - 19 -  Pentobarbital effects on a wide variety o f synaptic and non-synaptic targets might provide a window o f corticothalamic transmission for examining the mechanisms mentioned above.  1.2.11. Pre- and postsynaptic effects ofpentobarbital 1.2.11.1. Postsynaptic effects 1.2.11.1.1. Effects on receptor systems The most studied postsynaptic receptor target for pentobarbital action is the receptor for G A B A o f subtype A ( G A B A R ; Macdonald and Olsen, 1994). B y prolonging its decay, A  pentobarbital enhances the GABA-mediated CI" current, in a wide variety o f brain preparations, including ventrobasal thalamic neurons (Wan and Puil, 2002; Table 1.1). Pentobarbital can also directly activate CI" currents through G A B A A R (Mathers, 1987).  In  hippocampal neurons, pentobarbital has use-dependent postsynaptic  actions o f  promoting A M P A receptor desensitization (Jackson et al., 2003), which would contribute to corticothalamic S T D . Other studies have demonstrated an action o f pentobarbital to promote the desensitization o f the GluR2 subtype o f A M P A receptors (Taverna et al., 1994). The actions o f pentobarbital at this receptor subtype, also expressed i n thalamic neurons (Spreafico et al., 1994), are very sensitive to the actions o f cyclothiazide ( C T Z ) , a blocker o f A M P A receptor desensitization (Jackson et al., 2003).  - 20 -  Chapter 1. Introduction I Ran  Pentobarbital has depressant actions on N M D A receptor channels (Charlesworth et al., 1995). These actions include a reduction i n the probability o f channel opening, a shortening o f mean open time, and a decrease o f burst length.  1.2.11.1.2. Effects on non-receptor systems The postsynaptic effects o f pentobarbital on K currents have been studied i n cerebellar +  and hippocampal neurons (Carlen et al., 1985), and extensively studied i n ventrobasal thalamic neurons (Wan et al., 2003; Table 1.1). These effects include: 1) increasing the input conductance by activating a leak current, 2) activating a voltage-dependent K conductance, and 3) decreasing the hyperpolarization-activated N a / K +  +  +  inward current  (Ih).  Pentobarbital reduces C a  currents by increasing channel inactivation i n dissociated  spinal cord (Werz and Macdonald, 1985) and hippocampal neurons (ffrench-Mullen et al., 1993). These observations may explain pentobarbital effects i n decreasing lowthreshold spike firing i n thalamic neurons (cf. W a n and Puil, 2002).  1.2.11.2. Presynaptic effects of barbiturates Sherrington (1906) initially suggested that anesthetics reduce synaptic transmission rather than decreasing nerve conduction. Larrabee and Posternak (1952) showed that general anesthetics depress transmission through sympathetic ganglia, without effects on nerve conduction. Most investigations have dealt with the postsynaptic effects (Franks and Lieb, 1994). The contribution o f presynaptic components to barbiturate  anesthetic  Chapter 1. Introduction I Ran  - 21 -  properties has been neglected due to the difficulty i n recording from axon terminals and distinguishing between their pre- and postsynaptic effects. Only two reports indicated a reduction o f transmitter  release  without changes  have  i n action potential  configuration i n C N S neurons (Mathews and Quilliam, 1964; Weakly, 1969).  1.2.11.2.1. Effects on ion channels 1.2.11.2.1.1. Na channels +  A t anesthetic doses, barbiturates broaden action potentials at the frog neuromuscular junction (Thompson and Turkanis, 1973). The broadening o f the action potential might result from a hyperpolarizing shift i n activation (Wartenberg et al., 1999) or a usedependent block o f the N a channel (Rehberg et al., 1995). In principle, a broader action +  potential should promote transmitter release, by prolonging the depolarization o f the terminal and C a  influx.  1.2.11.2.1.2. Voltage gated Ca channels Ca  2 +  imaging studies show pentobarbital suppression o f C a  o f hippocampal neurons (Baudoux et a l , 2003).  2 +  entry into terminal branches  These observations suggest that  9-1-  pentobarbital enhancement o f C a promote S T D by reducing C a  1.2.11.2.1.3. K  +  channel inactivation (ffrench-Mullen et al., 1993) may  entry into the nerve terminals.  channels  channels are abundant at presynaptic terminals and are highly involved i n regulating  transmitter release (reviewed by Dodson and Forsythe, 2004). However, there is little  -22-  Chapter 1. Introduction I Ran  Table 1.1: Summary o f synaptic and non-synaptic actions o f pentobarbital Site  Effect  E D o or 5  IC50  Neuron  Reference  Receptor AMPA  NMDA  GABA  A  Glycine  use-dependent inhibition  20 | i M  Hippocampal (culture)  Decreased E P S P Amplitude  50 u M  Thalamocortical (slice)  Jackson et al., 2003  W a n et al., 2003  reduced mean channel open time  250 u M  prolonged current decay time  53 u M  Thalamocortical (slice)  increased mean channel open time  100 u M  Thalamocortical (slice)  W a n et al., 2003  prolonged current decay time  30 u M  Spinal dorsal horn (culture)  L u and X u , 2002  Olfactory (culture)  Charlesworth et al., 1995  W a n et a l , 2003  Ion channel Voltagedependent Na  Conduction block  3mM  +  Voltagedependent K  n  Increased  Iieak  Thalamocortical (slice)  W a n et al., 2003  8uM  Thalamocortical (slice)  W a n et al., 2003  Thalamocortical (slice)  W a n et al., 2003  +  Low-threshold no effect Ca' 2+ on h Voltagedependent C a 2+  Enhanced inactivation  Blaustein, 1968  8 pJVI  +  Voltageindependent K  decreased I , IKIR  Lobster (slice)  >100uM  3 |jM  Hippocampal (culture) ffrench-Mullen et al., 1993  Chapter 1. Introduction I Ran  - 23 -  direct evidence for anesthetics acting on K channels at nerve terminals. The function and +  properties o f K  +  channels vary widely at presynaptic locations. These include: 1)  dampening o f the presynaptic action potential by low-voltage-activated K channels; 2) +  faster repolarization (termination o f the action potential) by presynaptic high-voltageactivated K  +  channels; and, 3) activity-dependent modulation o f transmitter release by  interplay o f K - and Na -current activation. The structure o f presynaptic voltage-gated +  K  +  +  channels which mediate inactivating transient and non-inactivating delayed rectifier  currents  (IA  and  IKDR)  shows some similarity to K channels located i n the soma (Dodson +  and Forsythe 2004), suggesting possible interactions with pentobarbital.  1.2.11.2.2. Effects on the release machinery There are no reports on pentobarbital interactions with the soluble N-ethylmaleimidesensitive  factor  attachment protein receptors ( S N A R E )  proteins,  which  mediate  exocytotic transmitter release at active zones (cf. Duman and Forte 2003). However, imaging techniques demonstrated a pentobarbital effect o f suppression o f transient increases i n intracellular [ C a ] at axon terminals associated with a decrease i n the 2+  amplitude o f spontaneous EPSPs (Baudoux et al., 2003).  Since S N A R E proteins are  9-1-  sensitive to intracellular C a  alterations, one can assume an indirect effect that could  impair or increase release.  1.2.11.2.3. Effects on energy metabolism Pentobarbital impairs respiratory metabolism as well as glutamate synthesis, presumably at presynaptic terminals. Early studies have shown that pentobarbital reduces cellular  Chapter 1. Introduction I Ran  - 24 -  respiratory processes (Quastel and Wheatley, 1932; Aldridge and Parker, 1960; Chance and Hollunger, 1963), including neuronal glycolysis (Crane et al. 1978). The majority o f neuronal mitochondria are located at nerve terminals where A T P consumption is required for synthesis and release o f transmitters such as glutamate (Schwartz et al., 1979; Hertz and Zielke 2004). Hence, pentobarbital suppression o f metabolism produces a state o f energy deprivation, contingent on activity (Hertz et. al., 1986), which could eventually impair transmitter release.  1.2.11.2.4. Effects on transmitter release 1.2.11.2.4.1. Evoked release The presynaptic actions o f pentobarbital were initially studied i n spinal motoneurons (Weakly, 1969), and involve a reduction i n evoked transmitter release. Subanesthetic concentrations  o f pentobarbital decreased the number o f released quanta, without  changing the input resistance and firing threshold (quantal content; Figure 1.1). Hence, the study demonstrated for this site a presynaptic effect o f pentobarbital with a negligible postsynaptic effect.  In later studies, pentobarbital was found to enhance evoked transmitter release at the neuromuscular junction by increasing the number o f quanta released by nerve stimulation (Thomson and Turkanis, 1973; Seyama and Narahashi, 1975; Weakly and Proctor, 1977). The contrasting enhancement and reduction o f transmission were dose-dependent and attributable to the combined pre- and postsynaptic actions o f pentobarbital (Proctor and Weakly, 1976). The enhanced quantal release was assessed from the ratio o f evoked to  Chapter 1. Introduction I Ran  - 25 -  spontaneous end-plate potentials (EPPs), whereas postsynaptic effects were identified as decreased miniature amplitudes. Although initially the increased quanta was presumed to result from the increased duration o f a broader presynaptic action potential (Thompson and Turkanis, 1973), subsequent studies demonstrated no such effect (Weakly and Proctor, 1977). In summary, barbiturates have dose-dependent effects attributed to dual pre and postsynaptic actions that include both increase and decrease o f quantal release and reduction o f quantal size (Figure 1.2).  1.2.11.2.4.2. Spontaneous release Studies on spontaneous release provided greater detail about the complex nature o f barbiturate effects.  Barbiturate application increased the frequency and decreased the  amplitude o f spontaneous  acetylcholine release, independent o f extracellular [ C a ] 2+  (Pincus and Insler, 1978). These observations were quite similar to the  [Ca ]2+  independent enhancement o f miniature E P P frequency by ethanol (Quastel et al., 1971). These results implied that an effect o f barbiturates on C a  2 +  uptake determined the  magnitude o f the changes i n quantal content (Rahamimoff et al., 1976). In summary, barbiturates have opposing effects at the neuromuscular junction - a presynaptic action that increases miniature E P P frequency and quantal content and a postsynaptic action that increases membrane conductance, reducing amplitude o f spontaneous and evoked EPPs. Due to lack o f reliable methods o f assessment, the effects o f barbiturates on transmitter release have not received study at central synapses. A s investigated i n this thesis, the use o f fluctuation analysis can provide an accurate measure o f barbiturate modulation o f quantal parameters during short-term synaptic plasticity.  Chapter 1. Introduction I Ran  - 26 -  1.2.12. Theory of depletion model and fluctuation analysis Statistical estimation o f synaptic plasticity dates back to the work o f D e l Castillo and Katz (1954) who analyzed amplitude fluctuations o f spontaneous and evoked responses at the neuromuscular junction. The resemblance o f incremental amplitude fluctuation to the mean amplitude o f spontaneous miniature synaptic events formed the basis o f a 'quantal hypothesis o f transmitter release' (Del Castillo and Katz, 1954).  According to this  hypothesis, 3 parameters describe transmitter release at a given synapse: (1) the average amplitude o f the postsynaptic response (Q); (2) total number o f independent release sites at the synapse (N); and, (3) the average probability o f release across all sites (p). Changes i n p and Q constitute respectively the pre- and postsynaptic strength o f synaptic connectivity and either one or the other must be altered whenever there is plastic modification i n synaptic transmission. Alterations i n these parameters reflect a drug's mechanism o f action, e.g., a drug that acts exclusively at postsynaptic receptors must modify Q without an effect on p or N .  Since the classical approach to fluctuation analysis (del Castillo and Katz, 1954; B o y d and Martin, 1955) o f the synaptic response depends upon quantal content being low enough that there are many 'unit' responses, alternative statistical methods for analyzing quantal release, collectively called variance-mean analysis are often employed (Elmqvist and Quastel, 1965a; Vere-Jones, 1966; Clements, 2003).  The variance-mean analysis  uses the relation between the mean and the variance o f iterated synaptic responses.  The  binomial model o f transmission is valid i f the variance has a parabolic relation to the mean o f the response (Clements, 2003); the variance/mean ratio gives a measure o f Q  Chapter 1. Introduction I Ran  - 27 o  (Elmqvist and Quastel, 1965a,b; Clamann et a l , 1991; Silver et al., 1998; Scheuss and Neher, 2001). A major limitation o f the variance mean approach is that it is restricted to steady-state responses under stationary conditions o f release, i.e., all release sites are assumed to be independent and have the same release probability. When applied to long trains o f synaptic responses, the variance-mean analysis is useful for the study o f shortand long-term modification o f synaptic plasticity (Elmqvist and Quastel, 1965a).  The  present study utilized a modification o f the variance-mean analysis (see later i n this section).  The use o f the classical quantal analysis presents some difficulties i n interpreting changes in p, Q, and N . This method necessitates the use o f clearly detectable mean response amplitudes, plotted i n an amplitude histogram. In many preparations and various C N S neurons, however, the quanta are not easy to detect and do not form clear peaks i n the histogram. Such difficulty may result from sampling error or low signal to noise ratio. Additional factors that interfere with detection o f quantization include high quantal content and variability in quantal size.  In some preparations, quantal size is highly  variable i n the range o f 44-90%, as observed in distributions o f miniature excitatory postsynaptic currents (mEPSCs; Frerking and Wilson, 1996). The heterogeneity i n the probability o f transmitter release is i n the range o f 22-71% i n spinocerebellar tract neurons (Walmsley et al., 1988) and > 50% in hippocampal neurons (Murthy et al., 1997). The variability i n quantal size and release probability necessitate modifications o f the binomial model, such as the compound binomial, multinomial and compound  Chapter 1. Introduction I Ran  - 28 -  multinomial models (Brown et al., 1976; Redman, 1990; Quastel, 1997; Silver et al., 1998).  Despite limitations, the variance-mean method provides an independent approach for comparing the experimental means and variances with the model-based fits.  An  advantage is that changes i n synaptic parameters due to deviations from the simple assumptions o f the binomial model are reflected in the slope o f the linearized variancemean plot (Silver et al. 1998). The variance-mean method also is useful for analysis o f initial synaptic responses i n long trains at different  frequencies,  and at various  probabilities o f release due to systematic variations in the external [ C a ] . In cases where 2+  quantal content varies within the train, however, the variance-mean method, per se, cannot follow gradations o f the synaptic parameters (p and Q) within the train.  Another difficulty in the classical quantal hypothesis is the assumption that there is constant number o f participating release sites, defined as stationary. During repetitive stimulation, however, this assumption is not valid as the number o f participating sites is continuously altered and hence is non-stationary. Vere-Jones (1966) and Quastel (1997) pointed out an inherent variation o f N that occurs during release. Such variations can be estimated by using covariance analysis methods derived by Vere-Jones (1966), Quastel (1997), and Scheuss and Neher (2001) from the mathematical model o f Vere-Jones (1966). These methods essentially correct the variance/mean ratio for the effect o f p and provide, i n principle, estimates o f Q at successive responses i n iterated trains. The methods depend upon the validity o f the binomial model, which is indicated by the  Chapter 1. Introduction I Ran  - 29 -  existence o f negative correlations between responses to successive stimuli (Elmqvist and Quastel, 1965a; Scheuss and Neher, 2001). Assuming the maximal release o f one packet o f transmitter per stimulus per release site and little 'refill', a negative correlation between successive responses occurs because no release by one stimulus precludes release at the next (Vere-Jones, 1966). The correlation between successive stimuli might be influenced by presynaptic modulators o f release ( V i z i and Somogyi, 1989). A sudden increase i n the rate o f refill should show up as a positive interstimulus correlation (Matveev and Wang, 2000).  B y using the binomial depletion model discussed above, one can assess the effects o f barbiturates on quantal parameters o f transmission i n the thalamus during short-term alterations i n synaptic plasticity such as S T D .  1.3. Rationale W h y study S T D i n the thalamus? S T D contributes to the generation o f oscillations, an essential  behaviour  o f thalamic neurons  (Steriade  1999;  Castro-Alamancos and  Calcagnotto, 1999). For example, the effects o f S T D on thalamic firing behaviour depend on the composition, desensitization, and saturation o f postsynaptic receptors i n thalamic neurons (Chen et al., 2002; Bartlett and Smith, 2002).  There are no reports on the  presynaptic mechanisms that mediate short-term plasticity at corticothalamic synapses. Not surprisingly, most neuroscientists have assumed that thalamic plasticity is entirely postsynaptic  (Castro-Alamancos and Calcagnotto, 1999). This view has received  Chapter 1. Introduction I Ran  - 30 -  challenge by a demonstration o f a presynaptic form o f L T P at corticothalamic synapses (Castro-Alamancos and Calcagnotto, 1999). A frequency- and C a - dependence and 2 +  decreased paired-pulse facilitation characterized this form o f L T P .  However, the  previous study lacked a continuous measure o f the plastic alterations, which would emerge during longer stimulation train, en route to L T P .  Why  study the effects o f barbiturates on STD? A barbiturate-induced depletion o f  transmitter packets at nerve terminals might promote S T D during corticothalamic transmission. The best support for such a mechanism is based on the enhanced quantal release observed i n spinal motoneurons (Weakly, 1969). The increase i n released quanta depletes the store o f transmitter packets, which could exaggerate S T D .  The interactions o f barbiturates at the neuromuscular junction and on spinal motoneurons provide some rationale for studying the pre- and postsynaptic aspects o f corticothalamic transmission. Thalamocortical neurons in vivo are indeed sensitive because barbiturates disrupt their characteristic firing behaviour (Contreras and Steriade, 1996).  Current  theory maintains that these effects are postsynaptic and are due to enhanced G A B A e r g i c inputs.  In some studies (Castro-Alamancos and Calcagnotto, 1999), thalamocortical  neurons display postsynaptic mechanisms o f plasticity. Recent studies have shown that NMDA  receptors may enhance, whereas G A B A receptors may reduce the synaptic  strength o f corticothalamic responses. The plastic effects o f S T D would contribute to synaptic connectivity o f the thalamus during development (Bartlett and Smith, 2002). Both N M D A  and G A B A  receptors  are well-documented postsynaptic targets for  Chapter 1. Introduction I Ran  -31-  anesthetics but there is little or no information about potential presynaptic mechanisms that may explain the observed barbiturate-depression o f synaptic responses (Richards, 1971; Sawada and Yamamoto, 1985).  1.4 Objectives and research approach One o f the objectives o f the present study was to assess the changes i n quantal content and size, number o f release sites, and rate o f vesicular refill during repetitive stimulation, by using a corrected version o f the variance-mean method.  The correction o f the  variances was obtained using the covariances between successive synaptic stimuli (VereJones, 1966; Quastel, 1997; Scheuss and Neher, 2001). The present study examined how barbiturate anesthetics alter these corrected parameters. The hypothesis was that any effect o f pentobarbital on corticothalamic transmission, manifested i n a change o f S T D during repetitive stimulation, must be reflected i n changes o f quantal content and/or quantal amplitude (Q).  The study examined the anesthetic effects on synaptic transmission during repetitive stimulation o f corticothalamic axons, i n order to allow an expression o f short-term depression.  The binomial model was used to estimate changes i n synaptic parameters  such as quantal size and content, number o f release sites, and rate o f refill o f transmitter packets. These include S T D , observed as decreases i n amplitude ('rundown') o f the initial synaptic responses and increases in amplitude o f synaptic responses observed after a recovery from intra-train gap between stimuli. The findings enabled an assessment o f the  Chapter 1. Introduction I Ran  interactions  of  -32-  endogenously  released  glutamate  with  AMPA  differentiating pre- from postsynaptic mechanisms o f S T D .  receptors  while  B y pharmacological  inhibition o f A M P A receptor desensitization, it was possible to distinguish a postsynaptic contribution to S T D during repetitive stimulation.  The validity o f the covariance-corrected variance/mean method o f determining quantal amplitude was established i n control experiments which showed consistency with the binomial model - in particular, negative correlations between responses to successive stimuli that conformed with predictions o f the model. These investigations represent a determination o f anesthetic effects on both pre- and postsynaptic aspects o f excitatory synaptic transmission, for the first time i n neurons o f the C N S .  The anesthetic interactions with corticothalamic transmission presumably pertain to the mechanism o f barbiturate  anesthesia.  The investigations validated a method  estimating the pre- and postsynaptic contributions to synaptic plasticity.  for  Hence, the  present study obtained new knowledge about anesthetic interactions with the mechanisms o f plasticity, perhaps relevant to drug-induced amnesia as well as unconsciousness.  The effects o f pentobarbital on axonal conduction (Blaustein, 1968) and shunting o f thalamocortical neuron firing (Wan and Puil, 2002), suggested a presynaptic blockade o f action potential. Thus, it was worthwhile to compare the effects o f N a channel blockade +  with tetrodotoxin to those o f pentobarbital.  Chapter 1. Introduction I Ran  -33-  The high energy demand o f repetitive stimulation and the effects o f pentobarbital on metabolism (Quastel and Wheatley, 1932; Crane et al. 1978) provided rationale to examine whether conditions o f energy shortage would promote S T D . For this reason, STD  was  examined during conditions o f energy shortage imposed by glucose  deprivations.  The known depressant actions o f barbiturates provided rationale to examine whether pentobarbital could reduce the effects o f drugs that heighten excitability. The present studies investigated the effects o f pentobarbital on thalamic hyperexcitability due to extracellular spermine. First, it was necessary to examine whether extracellular spermine would heighten excitability by actions on membrane electrical properties, synaptic activation, and firing modes o f thalamic neurons.  Only a few investigations have  addressed this issue i n C N S neurons, finding that millimolar concentrations o f spermine depress population excitatory postsynaptic potentials (EPSPs) mediated by N M D A and non-NMDA-receptors,  as  well  as  inhibitory postsynaptic  potentials  (IPSPs) i n  hippocampal C A 1 neurons (DiScenna et al., 1994; Eterovic et al., 1997). Secondly, I examined the interactions o f pentobarbital with spermine for the presumed opposing actions o f the drugs on corticothalamic transmission, which modulated thalamic excitability.  The in vivo effects suggested that pentobarbital might promote oscillogenesis i n slice conditions. The present investigations also addressed pentobarbital effects on oscillatory behaviour i n the corticothalamocortical network o f neurons. In vitro oscillations were  Chapter 1. Introduction I Ran  - 34 -  induced b y corticothalamic stimulation i n combination with pentobarbital application at sub-  and anesthetic  concentrations.  Participating  receptors  were  identified  by  pharmacological blockade as well as surgical separation o f V B nuclei from the n R T . The frequency  distribution  o f pentobarbital  oscillations  was  determined  by  using  spectrocorrelograms, obtained for continuous extracellular multi-unit recordings. These investigations facilitated the identification o f a pro-oscillatory action o f a subanesthetic concentration o f pentobarbital on the C T C network.  1.5. Major questions Alterations i n corticothalamic transmission may have a crucial role i n oscillogenesis and modulatory mechanisms o f anesthesia. This thesis w i l l focus on actions o f pentobarbital on thalamocortical excitability, including the modification o f pre- and postsynaptic aspects o f synaptic transmission. The studies addressed the following questions: 1. Is pentobarbital capable o f inducing thalamic oscillations under slice conditions? 2. What receptors contribute to pentobarbital oscillations in vitro? 3. Does spermine enhance neuron excitability and does pentobarbital  modify  spermine action? 4. A r e the assumptions made by the binomial depletion model valid during S T D at corticothalamic synapses? Does the analysis reveal changes i n quantal parameters that are consistent with the binomial depletion model?  Chapter 1. Introduction I Ran  5. Does pentobarbital alter STD?  - 35 -  H o w do plastic alterations relate to changes i n  pre- and postsynaptic parameters? 6. During S T D , i n what ways are the actions o f pentobarbital similar to selective N a  +  channel blockade? Does pentobarbital affect energy metabolism? Does glucose deprivation mimic pentobarbital actions?  -36-  Chapter 2. Methods I Ran  Chapter 2 M E T H O D S  2.1. Slice preparation The A n i m a l Care Committee at The University o f British Columbia approved the procedures for these experiments. Experiments were performed on young adult SpragueDawley rats or gerbils (age 12-15 days) since they lack extensive myelination and are therefore ideal for proper formation o f patch-clamp seals.  Animals were decapitated  while under deep isoflurane anesthesia. The cerebral hemispheres were quickly removed (~1 min) from the cranial vault and immersed for 1-2 m i n i n ice-cold (0-2 °C) sucrose solution. The sucrose solution contained (in m M ) : sucrose, 248; NaHCC>3, 26; glucose, 10; K C 1 , 2.5; C a C l , 2; M g C l , 2; and N a H P 0 , 1.25. The brain was quickly transferred 2  2  2  4  to artificial cerebrospinal fluid ( A C S F ) , which had the same composition except for 124 m M N a C l instead o f sucrose. In repetitive stimulation experiments, glucose concentration was increased from 10 to 25 m M and the following drugs added (in m M ) : myo-inositol 3, Na-pyruvate 2, and ascorbic acid 0.4. This altered A C S F composition (320 mOsm) enhanced to the vitality o f repetitively stimulated neurons. The A C S F , on saturation with 95% O2 and 5% C O 2 , was adjusted to a p H o f 7.3-7.4. cube (-0.5  The brain was trimmed into a  cm ) containing the cortex and thalamus.  A Vibroslicer (Campden  Instruments, London, England) was used to cut 250 (im slices, for whole-cell recording, or 500 |om slices, for extracellular recordings. The slices were cut i n a hybrid coronal plane that formed a 45° angle with a sagittal plane. The slices used for extracellular recordings were somewhat thinner than similar slices described by Tancredi et al. (2000).  Chapter 2. Methods I Ran  - 37 -  The increased thickness (500 | j m compared to 250 Jim) facilitated the induction o f pentobarbital  oscillations in vitro. The use o f a hybrid cut slice maximized the  corticothalamic fiber content and enhanced the ability to evoke excitatory postsynaptic potentials  (EPSPs) or  currents (EPSCs).  In  a series o f extracellular  recording  experiments, a razor blade was used to surgically separate the ventrobasal ( V B ) nuclei from n R T .  The slices were electrically stimulated by using a bipolar tungsten electrode  (tip diameter - 1 0 0 itm), placed i n the slice at 0.2-0.3 m m from the recording electrode. For experiments performed in gerbils, horizontal slices were cut at 250 tun thickness that contained medial geniculate ( M G B ) and inferior collicular nuclei. In these slices, E P S P s were  evoked by stimulating at  a position mediodorsal  to the  M G B and  near  corticothalamic axons. Stimulation at this position resulted only i n EPSPs. The stimuli consisted o f single pulses o f approximately 30 V in amplitude (range, 10-100 V ) and 100-200 |is i n duration. The stimulation rate was 0.5 H z . Using these stimulus parameters, it was possible to evoke inhibitory postsynaptic potentials (IPSPs) when the electrode was placed i n the brachium, midway between the inferior colliculus and M G B . In experiments performed in rat thalamus, slices included portions o f the V B thalamus, internal capsule, and nucleus reticularis thalami (nRT). The postsynaptic potentials and currents were averaged and fitted with an a- function (pClamp 8 software), yielding the rise and decay time constants. Finally, the slices were incubated for 2-3 h i n A C S F at room temperature (22-25 °C), until required for recording, which was carried out at 2125°C.  Chapter 2. Methods I Ran  - 38 -  2.2. Drug application The slices were perfused on a nylon mesh with oxygenated A C S F and drugs. The drugs were prepared i n distilled water, firstly as stock solutions at - 1 0 0 0 times the required concentration and then frozen.  Just before the experiment, the stock solutions were  thawed and diluted in A C S F  for application.  triphosphate salt ( M g A T P ) , the C a AWA^-tetraacetic  acid  2 +  Pentobarbital, Mg -adenosine 5'2+  chelators, ethylene glycol-bis-(p-aminoethyl-ether)-  (EGTA)  or l,2-bis(2-aminophenoxy)ethane-/A^V /V'^V'r  tetraacetate ( B A P T A ) , Na -guanosine 5'-triphosphate (NaGTP), H E P E S , Q X - 3 1 4 , C s C l , +  D-2-amino-5-phosphono-valerate ( A P V ) , cyclothiazide (CTZ), kynurenate ( K Y N ) , and the inorganic chloride salts were obtained from Sigma (St. Louis, M O ) . The drugs were diluted i n A C S F and the p H adjusted in the range o f 7.3-7.4. Extracellular solutions were delivered i n two ways: 1) bath applications performed using a roller-type pump at a rate o f 2 ml/min through a submersion-type o f chamber with a volume o f ~ 0.3 m l , and 2) local  application by a glass pipette  (-100-200  (am tip diameter)  connected  by  polyethylene tubes to the various drug reservoirs. The local application approach allowed for a rapid switch (within < 5 s) between the various drugs (Quastel et al., 1971).  2.3. Extracellular recording  Extracellular multiunit activity was recorded i n lateral and medial portions o f ventral posterior thalamus o f submerged slices. The glass electrodes had tip diameters o f - 1 | i m and resistances o f 2-5 M Q when filled with 4 M N a C l . Aided by light microscopy (Zeiss Axioskop, Jena Germany), the electrode was positioned at 25-50 jam under the slice  Chapter 2. Methods I Ran  -39-  surface and recorded multiple unit potentials with an isolation amplifier (World Precision Instruments, Sarasota, F L , U S A ) . The signals were bandpass filtered with series, low and high-pass 8 pole Bessel filters (cutoffs, 1 and 16 H z ) . The signals were digitized at 5 k H z , and stored and analyzed (in part) with Axoclamp 8.2 software ( A x o n Instruments, Foster City, C A ) .  2.4. Whole-cell recording The electrical recordings were made i n the current- and voltage-clamp modes o f an Axoclamp 2 A amplifier ( A x o n Instruments, Foster City, C A ) .  A pClamp 8.2 software  ( A x o n Instruments) was used on a Pentium computer for data acquisition, storage and analysis. The voltage values were corrected for a measured junction potential o f -11 m V . For voltage-clamp recordings, the recording electrode were coated with Sylgard and the volume o f the bath solution lowered i n order to minimize electrode and bath capacitance, respectively. In current-clamp experiments, the intracellular patch solution contained (in m M ) : K-gluconate, 140; /V-2-hydroxyethylpiperazine-/V-2-ethanesulfonate ( H E P E S ) , 10; K C 1 , 5; N a C l , 4; adenosine 5-triphosphate (disodium salt), 3; guanosine 5-triphosphate (trisodium salt), 0.3; E G T A , 10; and C a C l , 1. This combination o f E G T A and C a  2 +  2  yielded a final [Ca ] o f 10 n M (calculated with M a x Chelator software). In experiments with B A P T A , E G T A was substituted with an equimolar concentration o f B A P T A , which yielded a final [ C a ] o f 1 n M . In voltage-clamp experiments, the patch solution 2+  contained (in m M ) :  Cs-gluconate 125; T E A - C 1 , 20; the lidocaine derivative Q X - 3 1 4 , 3;  H E P E S , 10; Na-phosphocreatine, 5; M g - A T P , 4; G T P , 0.4; and E G T A , 10, p H 7.3, 295-  Chapter 2. Methods I Ran  -40-  300 mOsm. The intracellular presence o f these drugs minimized the contribution o f postsynaptic N a , K , and C a +  +  2 +  channels while achieving an improved space clamp  (Cahalan and Aimers 1979; Konishi 1990; Budde et al. 1994).  2.5. Detection of signals 2.5.1. Direct method A n even number o f points (typically 4-10) were averaged around the point o f largest value (Figure 2.1). This approach reduced the error due to noise at the local maximum point. The amplitude o f the initial response was subtracted from baseline. Overlapping responses were obtained by subtraction o f the single exponential fit o f the late component from the preceding response. .  Highest point, highest noise  Figure 2.1: Direct method o f peak detection 2.5.2. Deconvolution  method  Another method for obtaining peak amplitude was deconvolution. The recorded signal represents the convolution o f the time course o f release o f multiple quanta by the time course o f a single quantum (Figure 2.2). Deconvolving the signal by the long x o f a quantum w i l l yield a value independent o f superposition on the tail o f a previous signal.  Chapter 2. Methods I Ran  If a = e~  l/T  -41-  where x is the time constant o f decay o f individual quanta,  then the  deconvolution o f a signal y; to produce: y j - yj-ayi_i/(l-a) excludes the components o f signal amplitude due to the 'tail' o f a previous signal. The resulting peaks are, however, much noisier than the original signal. Recorded signal  Deconvolved signal  time  Figure 2.2: Peak detection obtained by using deconvolution method.  2.5.3. First and second derivative method Peak were also detected by looking for zero crossings o f the first and second derivatives (Figure 2.3). In this method, the zero crossing o f the first derivative occurs at the location o f the peak. The second derivative crosses zero at the point o f maximal rise o f the signal. The two points were used to obtain a single exponential fit that contained the peak  Figure 2.3: First and second derivative method for peak detection. The first and second derivatives were used to detect the location o f the peak (1) and the maximal rate o f rise (2), respectively. The single exponential fit contained the peak estimate (1), above the zero crossing o f the first derivative (Chen and Regehr, 1999).  Chapter 2. Methods I Ran  -42-  Table 2.1: Comparison o f peak detection methods  Method Parameter  Direct  Deconvolution  1 and 2 derivative 1.23 ± 0 . 2 7  st  n d  Si (nA)  1.25 ± 0 . 3 5  1.25 ± 0 . 2 4  Sio (nA)  0.56 ± 0 . 1 1  0.53 ± 0.08  0.55 ± 0.09  Si/S  0.58 ± 0.08  0.56 ± 0.06  0.57 ± 0.09  1.35 ± 0 . 1 2  1.31 ± 0 . 1 3  1.34 ± 0 . 1 0  0.023 ± 0.007  0.021 ± 0.005  0.024 ± 0.009  -0.010 ± 0 . 0 0 3  -0.012 ± 0 . 0 0 4  - 0.009 ± 0.005  2  S12/S10 Var(Si) Cov(Si,S ) 2  Values are Mean ± S E M ; Si is E P S C amplitude where i corresponds to E P S C number V a r - variance, C o v - covariance (averages from 5 neurons)  Table 2.1 compares E P S C parameters obtained by the 3 peak detection methods. There were no significant differences between the 3 methods (P < 0.05, A N O V A test). Values obtained by the deconvolution method were normalized to the 1 peak value and rescaled st  by the mean amplitude obtained by the direct method. The direct and 1  st  and 2  n d  derivative methods did not require any scaling. Since a choice o f the peak detection method did not produce major errors i n parameter values, the direct method was used throughout the study, for the data presented i n the R E S U L T S section.  Chapter 2. Methods I Ran  - 43 -  2.6. Repetitive stimulation Excitatory post-synaptic potentials (EPSPs) or currents (EPSCs) were evoked by stimulating a small portion o f corticothalamic projections to V B thalamic neurons. For stimulation, a bipolar tungsten electrode was placed i n the internal capsule (IC). The IC was first stimulated with a strong (100V, 50-400 LIS) stimulus, the amplitude o f which was reduced as the electrode approached the surface o f the fibers. Once the electrode was just above the IC fibers the amplitude o f the stimulus was reduced to a value that was twice the minimal required to evoke E P S C s (or EPSPs). This indicated a contribution o f a small number o f stimulated fibers. Under these conditions o f stimulation, trains o f 20 stimuli were evoked at various frequencies (2.5-20 H z ) and statistical analysis performed using a binomial model.  2.7. Induction of plastic modifications of corticothalamic synaptic responses In early experiments, short-term depression was induced i n a long train o f repetitive stimulation (50 pulses) at 2.5, 5, 10, and 20 H z . Trains were applied with a 20 s intertrain interval to allow complete post-stimulation recovery. In most experiments, trains were 20 pulses; i n preliminary experiments, the time course o f post-stimulation recovery was assessed by applying trains with alternating inter-train intervals o f 1 and 10 s or more. Trains at the various frequencies were applied i n a sequence that included all possible combinations. This approach avoided biasing results by possible 'memory' i n the response from the previous frequency. For example, for frequencies 2.5, 5, 10 and 20 H z the sequence cycle was as follows: 10, 10, 20, 20, 5, 5, 2.5, 2.5, 10, 2.5, 10, 20, 5, 20, 2.5, 5. These stimulation protocols avoided time dependent correlations at a particular  Chapter 2. Methods I Ran  -44-  frequency. B y omitting the 1 1 stimulus in trains o f 20 stimuli, it was possible to assess th  the jump i n response that reflects more time for 'refill' i n the doubled gap between stimuli.  2.8. Analysis of extracellular recordings For multi-unit analysis, signal-to-noise ratio ( S N R ) was used for comparing the relative power density o f voltage fluctuations (Gabbiani and K o c h ,  1998).  Fast Fourier  Transforms were applied to filtered voltage traces, binned at 20 ms over 1-15 H z range. Dominant frequencies  were visualized with spectrocorrelograms  ( M A T L A B 6.1).  Autocorrelation functions ( M A T L A B 6.1) o f consecutive data sweeps (10 ms b i n width) were used to assess stationarity.  2.9. Fluctuation analysis of corticothalamic synaptic responses Amplitudes o f E P S C s at various locations i n the train were summarized in tables showing their descriptive parameters. Namely, the E P S C amplitudes o f the 1 (Si), 2 st  averaged responses 15  th  - 20  th  n d  (S2), and  (Plateau(Si5_2o)). The descriptive tables also contained  E P S C amplitude ratios early (S2/S1) i n the train, around the intra-train omitted stimulus (S12/S10), and between the plateau and the 1 response (Plateau/Si). st  The following equations were used to estimate the variance and covariance. F o r 2 successive E P S C s o f amplitude S; and Sj+i that are repeated r times:  -45-  Chapter 2. Methods I Ran  Var(S) = y (S -S ' r-\~l '' r  ) ll  var is calculated using sequentia pairs of repeats  2  , r + 1  -x cov(5,., S ) = — — £ fe, - S  cov is calculated using sequential pairs of repeats o f successive responses  r  M  t<r+l  r  \S  i+ir  - S  i+Ur+l  )/2  r = 1  Theory of corrected variance/mean as a measure of quantal  amplitude  Assuming a binomial behavior (Vere-Jones, 1966; Quastel, 1997): The release probability p is the product o f the output probability p , determined b y the 0  readily available pool, and the eligibility probability p , which depends on the rate o f A  refill and the stimulation frequency. Hence,  p =P  0  P A  where p is the probability for an output from an 'available' site and p is the probability 0  A  for a site to be available. Also,  _ m _mQ p - — - ~~ ~ N NQ  m - quantal content; N - number of release sites  V a r is expected to be: Var = m • Q • (1 - p) • (1 + CV ) 2  Between site variations  2  B  Var = m • Q • (1 - p) • (1 - p + CV ) 2  Within site variations  2  where C V - coefficient of variation The mean is always simply m-Q. W i t h predominantly between-site variation i n Q (Auger and Marty, 2000), the variance to mean ratio gives:  ^=eo c^).( -^)= .(i cr j).( -=) +  mean  1  e  N  +  1  1  *  NQ  Chapter 2. Methods I Ran  -46-  Consider now covariances. A t any one site i f there is no refill, i f there is a response at any one stimulus then there is none at the next, and vice versa cov(Sj, Sj) = < Sj Sj > - < S j > < Sj > cov(Sj, Sj) =  0  - < S; > < Sj >  while V a r (Sj) = < Sj >Q ( 1 + C V  2 Q  ) - < Sj >  2  For N sites, S's, Var's, and cov's are multiplied by N , hence V a r (SO = < Si > Q ( 1 + C V ) - < S; > / N 2  2  Q  cov (Si, Sj) = - < Sj > < Sj >/N  That is, cov (Si, Sj) multiplied by  < S; >/< Sj > is the same as the term i n V a r (Sj) that  involves N . Thus, one can define an apparent quantal size, Q', that is the same as i f quantal release were Poisson distributed, for each (average) member o f the train (Scheuss andNeher, 2001) "apparent quantal size" = Q' = Q ( 1 + C V ) = V a r (Sj) /< Sj > - cov ( S Sj)/ < Sj > 2  Q  i;  The same result is obtained i f variation o f quantal size is within sites. If refill (a) is appreciable, < Si-Sj > becomes positive and covariances become less negative with increased separation o f j from i (Vere-Jones, 1966; Quastel, 1997). A corollary is that correction o f variance/mean ratio to give Q' is incomplete, and the best one can do is use j = i + 1 for choosing which covariance to use.  Chapter 2. Methods I Ran  -47-  If, and only if, a covariance is non-zero, one can i n principle estimate N from any two responses:  < S >< S > N  =  -  cov  -—  valid only for cov(Sj, S,) < 0 in the absence o f refill  }  /  O  O  \  cov(S„Sj) cov(S„S,)  However, 1 / N ~ ~— is a derived number that should correspond to the N calculated from the decline i n calculated quantal contents o f responses i n trains. cov  It is notable also that i f there is nonstationarity o f Q between trains, e.g. i f local changes in conditions change 'shunting' between genesis and recording o f signals, one obtains + S,. (f )  Var(S ) =< S, > 0(1 +CV )(1 + f ) -  2  2  i  N (no refilD  cov(S ,S ) i  j  =  <  S  '  >  ^  S  j  >  +<S x t  5. >  (f)  where y is the between-train variance/mean which can be large relative to 1/N although 2  2  much less than 1 (Quastel, personal communication). The net result is that between-train variation can lead to a diminished, nulled, or positive covariance, obscuring the negative covariance due to the binomial nature o f the system. However, corrected variance-mean, Q', comes out the same as in the absence o f between-train non-stationarity. The same result is obtained with between-train non-stationarity o f N .  Estimation of quantal content ofsignals The number o f quanta, also referred to as the quantal content (m), was obtained separately from the initial five responses where E P S C s decline, and the subsequent  Chapter 2. Methods I Ran  - 48 -  plateau. From the 6 to 10 stimulus, the quantal contents were obtained from the ratio o f th  th  the size response to the corresponding quantal size:  'quantal content' = m, = <Si>/Q'j  where i > 10  The quantal contents o f the 1 response were obtained by dividing the response size by st  an average o f the quantal size at the first stimulus o f various frequencies (2.5, 5, 10, and 20 Hz). The quantal contents o f response 2-5 were obtained by dividing the response size by the quantal size averaged between the 2  n d  and 5  th  response.  In the absence o f refill, theoretical covariances are, for any pair o f outputs, i n terms o f S's, cov(Sj,Sj+i) = - <Sj>-<Sj+i>/N (see above), but with refill this becomes (Vere-Jones, 1966; Quastel, 1997):  cov (s ,Sj)= t  '—^— — J  + /(refill = a)  That is, the negative covariance becomes smaller the higher cc, the probability o f refill between stimuli. Evidently, this probability is greater the more stimuli are separated i n time, causing cov(Si,Sj)/(<S;><Sj>) to be most negative when j = i + 1, and progressively less negative with j = i + 2, i + 3, etc.  The presynaptic 'releasable store' and 'refill' According to the depletion model, signals fall in a train (STD) because each release o f quanta from a pre-existing store leaves the next stimulus with fewer quanta to draw upon. If release probability is constant, this model gives an exponential decline o f signals to a near steady state at which outputs balance refill o f the store. Because the data o f Elmqvist and Quastel (1965a) showed consistently higher S2's than expected on this basis, they  Chapter 2. Methods I Ran  - 49 -  therefore used a plot o f <S>  versus accumulated sum o f previous <S>'s to obtain b y  extrapolation a number representing a total 'apparent presynaptic store' presumably equivalent to Q N , 'refill' being apparently small early i n the train. This method was also used by Scheuss and Neher (2001). However, given negligible refill, one may also reason as follows: the first stimulus releases mi quanta leaving N - m\, the second m quanta, 2  leaving N - m\ - m , etc. Soon, few are left i f fractional release (P ) is more than ~ 0.3. 2  0  Then the release is P m u l t i p l i e d by ( N - mi - m2 - m^ ...etc.). The store (N) is therefore 0  m +m + m /P , or ra, + m + m +ra / P , or m, + m + w + m +ra / P etc. x  2  3  o  2  3  4  o  2  3  4  5  0  Therefore, given an estimate o f P , one has various estimates o f N , which become 0  overestimates, because there is some refill, the further one goes along the train. A fair compromise between underestimation o f N (at low P ) and overestimation (because o f 0  refill) is at  m, + m + m + m IP , or o f Q N , using S's instead o f w's, with P estimated 2  3  4  0  G  as 5, /(5 , + S + S + 5 ) o r S l(S + S + S ) 1  2  3  4  2  2  3  4  whichever is larger (Quastel, personal  communication), with the same assumption that Q's are constant. The result is less overestimation o f Q N , when refill (a) is not negligible, than with the method o f Elmqvist and Quastel (1965).  A still better estimate o f the 'releasable store' is Q N ' = QN/(1 + a ) and i n theory a is obtainable (Vere-Jones, 1966) since at equilibrium, S =QaP -N/(a+P -aP ) eq  0  0  0  while S =Q-P N X  0  giving P = S l{Q • N) 0  x  Chapter 2. Methods I Ran  Therefore,  -50-  defining  x = S I S = a I (a + P - a • P ), eq  i  o  rearranging  Q  gives  a = x-P l(\-x + xP ) g  0  However, this estimate is highly sensitive to error i n the estimate o f Q N , and therefore o f P and i f a is not small in the early part o f S T D w i l l result in an underestimate o f P and 0  0  a that cannot be corrected without information as to how P and a change during the 0  train. In the tables summarizing the data, Q N was used simply as a descriptive measure, equal to S + S + S + S 1p x  2  3  4  , with p being S  x  + S + 5 + S )or S /(S + S + 5 ) 2  3  4  2  2  3  4  whichever is larger.  The jump after an omitted stimulus In the plateau phase, outputs are the same. The number o f quanta present at stimulus j is mj and release is m = p • nj where p is fractional release. This leaves a store o f nj - p • n, y  and refill is a- ( N - nj • (1 - p)). Since the next store is the same, n  j + 1  =  n j  = - (1 - p) + a- ( N - n j - (1 -p)) n j  and rearranging, nj - nj- (1 - p) + a- nj- (1 - p) = a - N nj • (p + a - a-p) = a-N nj = a-N/(p + a - a-p) This expression, derived by Vere-Jones (1966), was used above to obtain a. N o w , i f stimulus j+1 is omitted, there is further filling, namely, a- ( N - nj).  Chapter 2. Methods  I Ran  -51-  After the gap, the store is n j+2 = rij + a- ( N - rij) = rij • (1 - a ) + a - N = rij • (1 - a ) + rij-(p + a - a-p) = nj-(l + p-a-p) O n the assumption that p does not change after the gap, m /mj = n'j+2/n'j = 1 + p- (1 - a ) j+2  2.10. Statistical comparisons A Student's Mest was used for comparing two groups and an analysis o f variance ( A N O V A ) test for comparing more than 2 groups. In some cases, a W i l c o x o n signed rank test was used for matched pairs o f groups. P < 0.05 was considered significant.  Chapter 3. Results I Ran  -52-  Chapter 3 RESULTS Parts o f the results i n the foregoing section have been published (Ran et al., 2004).  Part I. Pentobarbital oscillations in vitro in ventrobasal thalamus 3.1. Extracellular effects ofpentobarbital 3.1.1. Pentobarbital application and internal capsule stimulation Pentobarbital (PB) application (20 or 200 | i M ) did not produce oscillations i n 6 out o f 6 V B slices. Electrical stimulation o f the internal capsule also did not produce oscillations i n 12 out o f 12 slices.  Spontaneous oscillations were not observed during these  conditions. However, P B at 20 u M in combination with stimulation at 0.05 H z induced oscillations at 0.3-1 s intervals (Figure 3.1A) in all slices (n = 69).  The oscillations  typically lasted < 20 s from the stimulus onset and had a frequency distribution that remained  approximately  stationary  (Figure 3.IB), as confirmed by  autocorrelation  analysis.  Figure I C shows that P B increased the S N R near 3 and 8 H z , with smaller  increases near 11 and 13 H z . Since electrical stimulation was essential, P B application was a necessary, but not a sufficient condition for evoking oscillations. For convenience, these oscillatory responses w i l l be referred to as "PB-induced oscillations".  3.1.2. Effects of reduced extracellular Mg  2+  (fMg J ) 2+  e  Application o f P B increased the oscillations in slices made hyperexcitable i n low [ M g ] 2 +  media  (cf. Tancredi et al. 2000; Jacobsen et al. 2001).  Perfusion  (0.65 m M ) with, or without combined P B application did not  with low result  [Mg ] 2 +  e  i n oscillations  e  •53-  Chapter 3. Results I Ran  A  Control  PB 20 uM H^jv^^-jlH^---—Ind*—|t| Wash 150 uV 1 s  B  Control  .  PB (20 uM)  Wash  (  15 -  10  o c o BT 5  r  10  Time (min)  5 10 Frequency (Hz)  20  15  Figure 3.1. Pentobarbital induces extracellular oscillations i n ventrobasal nuclei during electrical stimulation o f internal capsule at 0.05 H z . A ) Pentobarbital (PB) reversibly evoked oscillatory discharge at 0.3-1 s intervals. B ) Spectrocorrelogram o f activity i n slice (A) illustrates that P B (horizontal bar) induced oscillations in the 1-13 H z range. The greyscale gradations correspond to the magnitude (black, highest; white, zero) o f oscillations as a function o f frequency. C) Signal-to-noise ( S N R ) is shown as a function o f frequency, averaged from n = 6 slices in control (open circles) and n = 6 slices bathed in 20 j i M P B (closed circles).  -54-  Chapter 3. Results I Ran  Low [Mg T 2  JWMfa  H'M  = T' M. i J '• •  n  i 'il  PB 20 uM  Wash 150 1s  B  low [Mg*]  PB (20 uM)  2  10  Time (min)  -i  Wash  20  r  5 10 Frequency (Hz)  15  Figure 3.2. Pentobarbital-induced oscillations in low M g medium. A ) Experiment similar to Figure 3.1 was conducted in a slice bathed i n 0.65 m M [ M g ] (low [ M g, 2 + n] ) Oscillations appeared in low [ M g ] during electrical stimulation. B ) P B application reversibly extended the frequency range o f firing from 5-10 H z , to 1-15 H z . C ) S N R versus frequency plots were averaged from 6 slices in low [ M g ] medium, before (open circles) and after 20 u M P B (closed circles). P < 0.001 in C . 2 +  2+  z+  e  2 +  e  2 +  e  Chapter 3. Results I Ran  - 55 -  in 6 out 6 slices (Figure 3.2).  Perfusion with low [ M g ] (0.65 m M ) and electrical 2 +  e  stimulation resulted i n oscillations at 5 to 9 H z i n 14 out o f 14 slices (cf. Figure 3.1). Under these conditions, P B application increased oscillatory activity i n 10 out o f 10 additional slices. Figure 3.2B shows that P B application reversibly intensified  the  oscillations and extended their frequency range from 5-9 H z , to 1-15 H z . Application o f P B increased the S N R predominantly near 8 H z , and to lesser extent near 3, 11, and 13 Hz  (Figure  3.2C).  PB-induced oscillations did not  apparently depend on  the  hyperexcitability due to low extracellular [ M g ] . 2+  e  3.1.3. Effects of raised temperature. It was necessary to determine i f the frequencies  o f PB-induced oscillations were  temperature dependent, as shown in vivo (Andersen and Andersen, 1968).  Spontaneous  oscillations were not observed in slices at 34 °C. Figure 3.3 illustrates a 10 °C increase i n temperature did not produce significant alterations i n the S N R over the 1-15 H z range (n = 6). Since the higher temperature did not significantly affect PB-induced oscillations, the study was conducted using slices maintained near 24 °C, promoting slice viability.  3.1.4. Application of high dose pentobarbital A 10-fold increase i n P B concentration induced oscillations which subsequently ceased, despite continuing P B application with internal capsule stimulation (n = 6). Figure 3.4 shows this biphasic effect for P B at 200 j i M .  The frequency range (1-15 H z ) at 3 m i n  was similar to that observed with P B at 20 | i M (Figure 3.4B).  The frequency range  Chapter 3. Results I Ran  -56-  PB 20 uM, 24 ° C  34°C 150 uV 1s  B  5 10 Frequency (Hz)  Figure 3.3. Effects o f raised temperature on pentobarbital oscillations. A n increase i n temperature from 24 °C to 34 °C did not alter the discharge frequency i n a slice (A) or the mean S N R (B) during oscillations induced by P B (20 u\M). Electrical stimulation (0.05 H z ) was applied throughout, as in Figure 3.1. n = 6, P > 0.05 i n B .  Chapter 3. Results I Ran  -57-  Figure 3.4. Time dependence o f effects o f increasing concentrations on pentobarbital oscillations. A ) A t 200 (i.M, P B application evoked oscillations i n a slice (24 °C) at 3 m i n which disappeared within 9 m i n o f the application. B ) S N R (n = 6) showed an increase at 3 m i n (closed circles) which disappeared within 9 min o f the application (open circles). Electrical stimulation (0.05 H z ) applied throughout, as i n Figure 3.1. P < 0.01 i n B.  Chapter 3. Results I Ran  - 58 -  narrowed to 1-4 H z before disappearance o f the oscillations at ~9 min.  The biphasic  effect o f P B at 200 u M occurred in 6 out o f 6 additional slices under low [ M g ] conditions and electrical stimulation (not shown). Hence, subsequent studies focused on 9+  the stable oscillations induced by P B at 20 | i M under normal [ M g ] conditions.  3.7.5. Effects of synaptic receptor blockade A possible involvement o f glutamate receptors i n PB-induced oscillations was examined, as found for the oscillations induced by electrical stimulation and low [ M g ] conditions 2+  (Tancredi et al., 2000; Jacobsen et al., 2001).  Application o f kynurenate (1 m M ) , an  ionotropic glutamate receptor blocker, reversibly abolished PB-induced oscillations i n 5 out o f 5 slices (not shown). Hence, glutamatergic transmission was essential for the P B induced oscillations. Experiments using applications o f G A B A antagonists showed that GABA-receptors likely participate i n PB-induced oscillations. During electrical stimulation, bicuculline methiodide (50 | j M ) reversibly induced oscillations at 1-4 H z (n = 3, not shown). comparison  with P B , co-application o f bicuculline with P B resulted  in  In  reduced  oscillogenesis at 5-15 H z i n 6 out o f 6 slices, without apparent changes i n the 1-4 H z oscillations (Figure 3.5A).  However, recent studies have shown that bicuculline  methiodide may have effects i n addition to G A B A  A  receptor blockade (Debarbieux et al.,  1998; Seutin et al., 1997) that could account for the depression o f oscillations.  Chapter 3. Results I Ran  - 59 -  The question o f receptor specificity was further examined by applying another G A B A A antagonist, gabazine (Uchida et al.,  1996;  Seutin et al.,  1997).  Application o f gabazine  (20 L I M ) reversibly induced oscillations at 1-4 H z in 6 out o f 6 slices, similar to bicuculline (not shown).  In comparison with P B , co-application o f gabazine (20 j i M )  with P B resulted i n reduced oscillogenesis at 5 - 1 5 H z i n 6 out o f 6 slices, without apparent changes i n oscillations at  H z (Figure  1-4  3.5B).  Therefore, it seemed likely that  gabazine- and bicuculline-sensitive G A B A A receptor interactions participated i n P B induced oscillations at frequencies above 4 H z .  Application o f the G A B A B receptor antagonist, C G P 35348 (100 n M ) during electrical stimulation, reversibly induced oscillations at 5-15 H z i n 3 slices (not shown).  Co-  application o f C G P 35348 with P B resulted in reduced oscillogenesis at 1-4 H z and 11-15 H z , sparing the 5 to 10 H z range i n 6 out o f 6 slices (Figure 3.5C).  Thus,  GABAB  antagonism induced oscillations centred near 8 H z , and during P B application, resulted i n suppressed oscillations at lower and higher frequencies.  Combined antagonism by bicuculline and C G P 35348 did not evoke oscillations during electrical stimulation i n 5 slices (not shown). Unexpectedly, oscillations i n the 5-10 H z range persisted during co-application o f G A B A receptor antagonists with P B i n 6 out o f 6 slices (Figure 3.5D). Hence, G A B A  A  and G A B A  B  mediated inhibition was apparently  not an absolute requirement for oscillations during P B application.  -60-  Chapter 3. Results I Ran  B  C  Frequency (Hz)  Frequency (Hz)  Figure 3.5 Antagonists o f G A B A , and glycine receptors modulate frequency o f pentobarbital oscillations. A - F (n = 6 i n each panel) show mean S N R as a function o f frequency during application o f 20 u\M P B . A ) Bicuculline (BIC, 50 | i M ) decreased the S N R o f the oscillations at middle, and especially at high frequencies (P < 0.01). B ) Gabazine ( G B Z , 20 | j M ) also decreased the S N R o f the oscillations i n the middle and high frequencies, similar to bicuculline in A (P < 0.01). C ) C G P 35348 ( C G P , 100 u M ) decreased the S N R o f the oscillations at low and high frequencies (P < 0.01). D ) Combined application o f B I C and C G P 35348 did not eliminate P B enhancement o f the S N R i n the middle frequency range (P < 0.01). E) Strychnine (STR; 1 ixM) decreased the S N R o f the oscillations at low and high frequencies, similar to C G P 35348 application (P < 0.01). F) S T R (1 nM).combined with B I C and C G P 35348 abolished P B enhancement o f S N R (P < 0.01). In A - F , the S N R for control P B oscillations is shown as a dotted reference plot. Horizontal line, S N R = 1.  Chapter 3. Results 1 Ran  - 61 -  In view o f the incomplete blockade o f all oscillation frequencies by G A B A receptor antagonism, it was necessary to apply picrotoxinin, which blocks at a different site on the G A B A A receptor than bicuculline or gabazine, as well as inhibiting homomeric glycine receptors ( Y o o n et al., 1998). Picrotoxinin (50 (iM) during electrical stimulation did not evoke oscillations in 6 out o f 6 slices. Application o f picrotoxinin (50 u M ) (n = 4), alone, or co-applied with C G P 35348 (n = 2), eliminated PB-induced oscillations (not shown). These results are consistent with picrotoxinin blockade o f G A B A A and glycine receptors.  The next experiment determined the effects o f strychnine, a glycine-receptor antagonist, on PB-induced oscillations. B y itself, strychnine (1 u.M) induced oscillations, but not during G A B A receptor antagonism. investigations.  These effects were not studied further i n these  During P B application, strychnine (1 L I M ) reversibly decreased the  oscillations at low and high frequencies, sparing those at 5-10 H z i n 5 out o f 5 slices (Figure 3.5E). This effect was similar to that o f C G P 35348. antagonism,  co-application  o f strychnine  with P B resulted  During G A B A receptor only i n  rudimentary  oscillations i n 5 out o f 5 slices (Figure 3.5F). Hence, both G A B A and glycine receptors apparently modulated PB-induced oscillations.  3.1.6. Pentobarbital-induced oscillations in separated thalamic nuclei. The possibility that G A B A e r g i c connections from n R T were necessary for PB-induced oscillations was examined by studying the effects o f P B i n V B nuclei and n R T , isolated from each other (Figure 3.6).  In V B nuclei surgically isolated from n R T (Figure 3.7A),  P B application evoked oscillations at > 5 H z during electrical stimulation at a V B  Chapter 3. Results I Ran  site that was 1-2.5 m m from the recording electrode (Figure 3.6).  -62-  G A B A receptor  blockade by co-applied bicuculline and C G P 35348 had no significant effect on the oscillations (not shown, n = 5). Hence, receptors for G A B A did not appear to have a modulatory role i n V B nuclei, isolated from n R T .  In view o f the persisting oscillations during G A B A receptor blockade, the effects o f strychnine i n V B nuclei were determined, after isolation from n R T . Strychnine (1 p M ) reversibly eliminated PB-induced oscillations (Figure 3.7; n = 6). This implied that P B actions on glycine receptor systems were essential for the oscillations i n V B nuclei, deprived o f G A B A e r g i c inputs.  Since disconnection from n R T altered the frequency o f oscillations i n V B nuclei, the effects o f P B i n n R T were determined, before and after its disconnection from V B nuclei. Before disconnection, P B application (20 p M ) induced oscillations at 1-12 H z i n n R T during electrical stimulation o f the internal capsule i n 5 out o f 5 slices (1-10 H z i n Figure 3.8). Surgical disconnection from V B nuclei i n these 5 slices did not significantly affect the ability o f P B application to induce n R T oscillations i n a similar frequency range (Figure 3.8). Hence, P B can induce oscillations in n R T , at a site distinct from V B nuclei.  G A B A receptor blockade by co-applied bicuculline and C G P 35348 abolished the oscillations induced by P B i n n R T (not shown). This occurred with (n = 6), or without  Chapter 3. Results I Ran  -63-  Figure 3.6. Photomicrograph o f sagittal slice shows complete separation (asterisk) o f V B nuclei from n R T . C x , cortex; Hipp, hippocampus; IC, internal capsule; n R T , nucleus reticularis thalami; Po, posterior thalamic nucleus. Vertical arrows: rostral, R ; caudal, C .  Chapter 3. Results I Ran  -64-  Isolated VB (no nRT) PB  PB+STR  5  10  15  Frequency (Hz)  Figure 3.7. Pentobarbital oscillations i n electrically stimulated V B nuclei, after surgical separation from n R T . Top: In a slice (top and middle), P B (20 p M ) evoked 6-15 H z oscillations i n V B nuclei, after separation from n R T . Middle: Co-application with S T R (1 ixM) abolished these oscillations. Bottom: Plot of S N R as a function o f frequency quantifies the effects o f S T R in 11 slices (P < 0.01, Mest).  Chapter 3. Results I Ran  -65-  R e c o r d i n g in n R T  0  10  Time(min)  20  30  0  10  Time(min)  20  30  Figure 3.8. Pentobarbital oscillations in electrically stimulated n R T , before and after its surgical separation from V B nuclei. A ) P B (20 p M ) induced oscillations at 1-10 H z i n n R T during stimulation o f the internal capsule. B ) in a another slice, P B (20 p M ) induced oscillations at 1-9 H z in n R T , electrically stimulated at 0.05 H z after surgical separation from dorsal thalamic nuclei.  Chapter 3. Results I Ran  - 66 -  (n = 6) connections to V B nuclei. Strychnine application had no significant effect on the oscillations induced by P B i n n R T , with (n = 5) or without (n = 5) connections to V B nuclei (not shown).  Hence, P B can induce purely G A B A e r g i c oscillations i n n R T ,  separated from V B nuclei.  3.1.7. Discussion Pentobarbital oscillations required ionotropic glutamate excitation, but not elevation o f temperature from 2 4 ° to 34° or low [ M g ] conditions. Although they can occur 2+  spontaneously under different conditions (Jacobsen et al., 2001), oscillations were never observed without electrical stimulation, i n the present study.  The oscillations had a  broader frequency range than seen with low extracellular [ M g ] . Hence, pentobarbital 2+  likely acted on M g  independent sites.  Receptors for G A B A modulated pentobarbital-induced oscillations. G A B A A antagonism with bicuculline methiodide or gabazine eliminated oscillations at 10-15 H z , but not i n the lower frequency range. Bicuculline and gabazine had equivalent effects, with and without co-applied pentobarbital. The modulation o f pentobarbital-induced oscillations was not likely due to unselective actions o f bicuculline methiodide (cf. Debarbieux et al., 1998). Gabazine blocks G A B A A receptors (Uchida et al., 1996) without producing the effects o f bicuculline methiodide on intrinsic membrane currents i n C N S  neurons (Seutin  et al., 1997). The above results imply that G A B A A receptors modulated the oscillations i n the high frequency range.  Chapter 3. Results I Ran  - 67 -  Picrotoxinin abolished oscillations induced by pentobarbital, i n marked contrast to the other G A B A A antagonists. Picrotoxinin itself did not induce oscillations, i n agreement with previous reports (Jacobsen et al., 2001). In addition to blocking G A B A A receptors, picrotoxinin inhibits homomeric glycine receptors  (Yoon et al., 1998). Thus, a  picrotoxinin-blockade o f both G A B A A and glycine receptors apparently mimicked the effects  o f co-applied strychnine  and bicuculline. Apparently, G A B A  B  receptors  modulated the oscillations i n both low and high frequency ranges. The oscillations persisted in the 5-10 H z range during C G P 35348 (Jacobsen et al., 2001 and Ziakopoulos et al., 2000) or strychnine applications.  Unexpectedly, G A B A receptor blockade did not abolish the oscillations, rather they persisted i n a 5-10 H z range. This reflected an altered network function because pentobarbital-induced oscillations in the same frequency range i n ventrobasal nuclei deprived o f reticular G A B A e r g i c inputs. After this surgical isolation, or during G A B A receptor  blockade,  strychnine  application  eliminated  the  oscillations.  In  nRT  disconnected from ventrobasal nuclei, strychnine did not affect the oscillations mediated by glutamate and G A B A . In summary, pentobarbital induced oscillations i n isolated networks o f the ventrobasal and reticular nuclei, mediated by glutamate receptors and modulated by overlapping interactions at G A B A , G A B A , and glycine receptors. A  B  Chapter 3. Results I Ran  - 68 -  Parts o f the results i n the following section have been published (Ran et al., 2003).  Part II. Pentobarbital modulation of N M D A receptors in corticothalamic transmission The present section o f the thesis addressed the issue o f how modulation o f N M D A receptors affects the excitability o f thalamic neurons. N M D A receptors are located at distal synapses on thalamic neurons and receive extensive cortical inputs. Abnormal modulation o f N M D A  receptors may result i n thalamic hyperexcitability, which  contributes to some forms o f epilepsy. A depressant action o f pentobarbital may reduce the effects caused by such abnormal modulation. The following experiments examined how  pentobarbital affects the modulation o f thalamic excitability by spermine, an  endogenous polyamine with extracellular modulatory effects on N M D A receptors.  3.2.1 Effects of spermine 3.2.1.1. Tonic firing  Spermine application reversibly increased the number o f action potentials i n all neurons depolarized from rest by current pulse injection. Spermine (100 |JM) applied for 3 - 6 m i n induced tonic firing o f action potentials on top o f subthreshold responses. When action potentials were present i n the control, spermine application increased the rate o f firing (Figure 3.9A). Long recovery times o f 35 - 45 m i n characterized spermine's effects on thalamic firing modes after 6 m i n applications. In the neuron o f Figure 3.9A, substantial recovery occurred at ~ 32 m i n after discontinuing the spermine application.  Chapter 3. Results I Ran  -69-  The spermine-induced increase i n the firing frequency was concentration-dependent over the range o f 5 0 and 5 0 0 J J M (n = 1 9 , Figure 3 . 9 B ) . In addition, application o f 1 | i M spermine did not affect firing (n = 2); however, at 1 m M , there was a marked increase i n the firing rate (n = 2), without any apparent recovery (data not shown). Spermine, applied at an ED50 o f 1 0 0 L L M (cf. Figure 3 . 9 B ) , reversibly increased the number o f action potentials per pulse by an average o f ~ 8 0 % i n nine neurons (control, 1.8 ± 0 . 3 action potentials/pulse and spermine application, 3 . 3 ± 0 . 4 action potentials/pulse, paired t-test, P<0.01).  3.2.1.2. Passive membrane properties  The increased firing due to spermine did not likely result from changes i n the passive membrane properties which did not greatly change during 3 to 6 m i n spermine applications (cf. subthreshold responses in Figures. 3.9A and 3.11 A ) . The average resting potentials were -67 ± 4 m V during the control period, and -66 ± 5 m V during applications o f spermine at 50 - 500 \iM (n = 19). Spermine application did not significantly change the mean membrane time constant ( x = 64 ± 6 ms i n control, and 76 ± 6 ms during m  spermine application, paired Mest) and mean input resistance (Rf= 772  ± 3 8 M Q in  control, and 756 ± 6 1 M Q during spermine application, paired Mest), computed from the responses to hyperpolarizing current pulse injections in 19 neurons held at -65 m V . The lack o f effect on passive membrane properties may result from spermine actions on distal dendrites. Hence, the spermine-induced effects on the passive properties could not account for the changes i n firing threshold.  Chapter 3. Results 1 Ran  -70-  Figure 3.9. Spermine enhanced tonic firing i n a concentration-dependent manner i n M G B neurons. A ) Spermine application (100 L I M , 3 min) enhanced action potential firing evoked by current pulses (25 and 50 p A , 500 ms, 1.5><threshold). Holding potential, - 65 m V . Vertical upper bar, 30 m V and lower bar, 60 p A ; horizontal bar, 150 ms. B ) Increase in number o f action potentials per pulse was concentration dependent. The control firing was 1.8 ± 0.3 action potentials/pulse (n =9). The ED50 was - 1 0 0 L I M for spermineenhanced firing which approached saturation at 200 j i M .  Chapter 3. Results I Ran  -71-  3.2.1.3. Action potential threshold Spermine (100 u M ) decreased the latency to tonic firing by decreasing the threshold (Figure 3.10). Spermine decreased the firing threshold from -51.0 ± 0.6 m V to -57.1 ± 2.2 m V (Figure 3.10B). Significant changes i n action potential amplitude did not accompany the decreased threshold. Figure 3.10B summarizes the effects o f spermine on firing threshold for six neurons.  The possibility that N M D A receptors mediated the effects on the firing threshold and tonic firing rate was examined by determining the interactions o f spermine and the competitive antagonist, A P V (50 piM). A s shown in Figure 3.10B, spermine reduced the threshold voltage for an action potential evoked with a 500 ms current pulse, b y an average o f 6.2 ± 1.1 m V . O n recovery from spermine (washout, Figure 3.10B), application o f A P V alone, or in combination with spermine, did not significantly change the firing threshold (during A P V , - 52.1 ± 1.9 m V and A P V + spermine, -53.0 ± 1.7 m V ; n = 6) or changes i n membrane properties that could account for the blockade o f the spermine-induced reduction i n firing threshold. This signified that A P V acted on N M D A receptors to completely block spermine action. Since these neurons had received a spermine application prior to A P V , A P V was also applied to five neurons that had not previously received a drug application i n order to assess the possibility o f constitutive release o f glutamate i n the slice. Here, A P V produced an increase i n threshold, which remained largely unaltered by a subsequent, combined application with spermine (Figure 3.10C). A l l neurons showed substantial recovery at 15 m i n after discontinuing the  Chapter 3. Results I Ran  -72-  A J  Control Spermine Washout  APV  +APV  Washout  + APV  Figure 3.10. Spermine (100 p M ) decreased the action potential threshold. ( A ) Spermine application produced a leftward shift i n action potential latency (current pulse duration, 500 ms). Arrows point to threshold in control and spermine. (B) N M D A receptors mediated spermine effects on action potential threshold. The control threshold was -50.9 ± 0.6 m V , which spermine reduced to -57.1 ± 2.2 m V (n = 6). Partial recovery was observed after 15 m i n (-52.4 ± 0.6). Blockade o f N M D A receptors by A P V (50 p M ) reduced the threshold by < 1 m V . A reduction i n threshold was not observed during coapplication o f A P V and spermine (-0.9 ± 0.6 m V , n = 6). (C) A P V (50 p M ) increased firing threshold i n five neurons from -52.3 ± 0.7 to -48.7 ± 0.5 m V . Subsequent spermine application did not alter the increased threshold (-49.1 ± 0.6 m V ) . Two-way A N O V A ; * in (B) indicates P < 0.01 and i n (C) P < 0.05. Vertical bar i n (A), 15 m V ; horizontal bar, 50 ms.  Chapter 3. Results I Ran  -73-  application. These data implicated an N M D A receptor mechanism i n the spermineinduced decrease i n the threshold.  N M D A receptors also mediated the spermine-induced increase i n firing rate. In the neuron o f Figure 3.11 A , spermine application (100 ( i M , 3 min) reversibly increased the number o f action potentials during a 500 ms current pulse injection from one action potential i n the initial control, to three action potentials. In the neurons that had not previously received a spermine application, an increase i n the current pulse amplitude during action potential blockade due to A P V application, produced a return o f the action potential (Figure 3.1 IB). The A P V blockade o f spermine-induced increase i n firing also was overcome by an increase i n the current amplitude. The A P V - i n d u c e d blockades o f action potentials and spermine enhancement  o f firing were not attributable to an  increased input conductance and were completely reversible. The graph o f Figure 3.1 I C summarizes the data that implicated N M D A receptor mediation.  It was necessary to examine the possibility that n o n - N M D A receptors for glutamate contributed to the increased firing during spermine application. These studies determined the interactions o f spermine with an A M P A receptor antagonist, C N Q X . Application o f C N Q X (30 f i M ) for 6 m i n did not result i n significant changes in evoked action potential firing, configuration, or membrane electrical properties. Subsequently, combined C N Q X and spermine application did not greatly alter the reduction in threshold and ~ 200% increase i n firing rate evoked by current pulses (amplitude ~1.5 x threshold), as observed with prior spermine application i n all five neurons ( C N Q X , 1.4 ± 0.3 action potentials per  Chapter 3. Results 1 Ran  -74-  Control  Spermine  APV  APV+ Spermine  Washout  Washout ,  Figure 3.11. Spermine increased tonic firing by interacting with N M D A receptors. ( A ) Spermine application (100 L I M , 3 min) reversibly induced firing. After a 15 m i n washout from spermine, A P V (50 | i M , 6 min), an N M D A receptor antagonist, blocked the evoked action potential. Firing was not observed during co-application o f A P V and spermine (3 min). Washout shows recovery at 10 m i n after discontinuing the co-application. Lower traces show hyperpolarizing tests for input resistance. (B) Application o f A P V (50 u M , 6 min) abolished firing induced by just-threshold current pulse (40 p A ) . A subsequent 3 min co-application o f spermine and A P V did not alter this suppression (lower superimposed traces i n middle panel). A two-fold increase i n current amplitude overcame the blockade during A P V application, alone, or during co-application with spermine (upper superimposed traces i n middle panel). Recovery was observed after 10 m i n washout. (C) Summary o f spermine effects on firing i n six neurons. A N O V A ; *P < 0.01 - significantly different from control, **P < 0.05 - significantly different from spermine. Vertical bar, 30 m V ; horizontal bar, 200 ms.  Chapter 3. Results I Ran  -75-  pulse, and CNQX+spermine, 4.2 ± 0.4 action potentials/pulse; data not shown). Hence, the increase i n tonic firing rate due to spermine application did not likely involve A M P A receptor interactions.  3.2.1.4. Membrane rectification The following experiments examined the possibility that the spermine-induced increase in  tonic  firing  involved voltage-dependent  membrane  properties.  For  example,  thalamocortical neurons exhibit larger voltage responses to depolarizing, compared to hyperpolarizing current pulses (Tennigkeit et al., 1996; Parri and Crunelli, 1998; cf. Figure 3.12B). Spermine (100 p M ) application for 3 m i n increased inward rectification i n a range between the rest and firing threshold, but did not appreciably change the responses at hyperpolarized potentials, down to - 100 m V (n =19; Figure 3.12A). Quantification o f the increase i n rectification on depolarization was difficult because spermine application shortened the latency to firing (cf. arrows i n Figures. 3.1 OA and 3.12A). Application o f A P V (50 p M , 6 min) completely blocked the rectification i n the upper right quadrant o f the voltage - current (V - 1) relationship. A subsequent coapplication with spermine (100 p M ) did not greatly change this curve. The graph o f Figure 3.12 A (right) summarizes these findings for six neurons.  There was little or no involvement o f A M P A receptors in the spermine-induced (100 p M , 3 min) enhancement o f rectification produced by depolarizing current pulses. C N Q X (30 p M , 6 min) did not alter spermine's effects on the rectification i n five neurons. The average voltage response during co-application o f C N Q X and spermine (15.9 ± 0.6 m V )  Chapter 3. Results I Ran  - 76 -  was significantly different from control (11.5 + 0.5 m V ) or C N Q X application (11.3 ± 0.4 m V ; A N O V A , P < 0.05). Hence, the spermine-induced increase i n the depolarizing responses involved N M D A receptors, but likely not A M P A receptors.  The next investigations examined whether spermine increased the rectification i n the upper right quadrant o f the V - I relationship by interacting with a persistent N a  +  conductance. The rectification observed on depolarization from — 7 0 m V to threshold involved a persistent N a conductance, sensitive to T T X blockade (Tennigkeit et al., +  1996; Parri and Crunelli, 1998). Blockade o f voltage-dependent N a channels with T T X +  (0.6 p M , 6 min) decreased the slope o f the V -1 relationship, more i n the depolarizing quadrant than i n the hyperpolarizing quadrant. The blockade with T T X nullified the ability o f spermine (100 p M , 3 min) to increase rectification on depolarization (n = 6 ; Figure 3.12B). The results imply that spermine increased rectification i n the upper right quadrant o f the V -1 relationship by increasing a TTX-sensitive, voltage-dependent N a  +  conductance.  The spermine-induced enhancement o f rectification on depolarization o f the neuron also 2+  may depend on extra or intracellular C a , as i n neocortical neurons (Crill, 1996). Hence, the  spermine-induced  enhancement o f voltage  responses to  depolarizing  current  injections were measured during intracellular application o f B A P T A (10 m M ) and 2+  extracellular perfusion with nominally C a - free A C S F . In the neuron o f Figure 3.13A, 2+  perfusion o f C a  - free A C S F did not greatly alter these depolarizing responses. In four  neurons, a 50 p A current pulse evoked average responses o f 9.8 ± 0.6 m V i n control  Chapter 3. Results I Ran  -77-  Figure 3.12. Effects o f spermine (100 | J M , 3 min) on membrane rectification. ( A ) Voltage -current (V - I) relationship o f a neuron shows that spermine increased depolarizing response which was abolished during combined application (3 min) with A P V . A P V (50 L I M , 6 min), alone, reduced rectification i n upper right quadrant. V -1 curve after 15 m i n washout shows substantial recovery. Graph at right summarizes effects o f spermine, A P V , and their co-application on rectification. The response on depolarization increased from 12.5 ± 0.5 m V (control) to 17.5 ± 0.8 m V during spermine application, and after 15 m i n washout, recovered to 12.9 ± 0.7 m V . Subsequent A P V application reduced the response on depolarization from 12.9 ± 0.7 m V (first washout) to 9.2 ± 0.4 m V ( A P V ) . Co-applied A P V and spermine did not greatly alter rectification (8.7 ± 0.3 m V ) . Recovery from A P V and spermine occurred after 15 m i n (12.7 ± 0.5 m V ) . Holding potential, -70 m V . (B) V - I relationship for a neuron shows that T T X application (0.6 u M , 6 min) decreased rectification on depolarization over a ~10 m V range. Co-applied with T T X , spermine did not alter rectification i n upper right quadrant. A 20 p A pulse was sufficient to observe rectification on depolarization, whereas a - 50 p A pulse produced little or no rectification on hyperpolarization. Graph at right summarizes effects o f T T X and spermine on rectification. Rectification on depolarization decreased from 13.1 ± 0 . 8 m V (control) to 10.2 ± 0 . 8 m V during T T X application. A subsequent co-application with spermine did not greatly alter depolarizing responses (10.8 ± 0.7 m V ) . Inserts i n upper left quadrants o f (A) and (B) show superimposed responses (7 m V ) to depolarizing and hyperpolarizing current pulses (duration 500 ms) o f 60 and - 60 p A , during control (C), spermine (S), and at 3 m i n o f co-application o f T T X and spermine (TTX+S). Bar graph values are mean ± S . E . M . A N O V A test, n = 6; * and **P < 0.05.  Chapter 3. Results 1 Ran  - 78 -  2+  A C S F and 10.0 ± 0.5 m V i n 0 m M [Ca ]. The response increased to 15.2 ± 1 m V during 2+  spermine  application (in 2 m M C a  A C S F ; A N O V A , P < 0.01) which did not 2+  significantly change during combined application o f spermine and 0 m M [Ca ] (10.2. ± 0.3 m V , n = 4 , P > 0.05, A N O V A ) . 2+  In contrast, the intracellular application o f B A P T A , a more rapid C a  chelator than  E G T A , eliminated the spermine-induced enhancement o f voltage responses, observed on depolarization (Figure 3.13B). In neurons recorded with B A P T A - c o n t a i n i n g pipettes, spermine application did not alter the responses to depolarizing currents (average o f 12.4 ± 1 m V i n control and 12.4 ± 0.7 during spermine ; n = 4). This implied that spermine 2+  induced either C a  2 +  entry into the neuron or a C a  -dependent conductance which  enhanced the subthreshold depolarizing responses and promoted rectification i n the upper right quadrant o f the V-1 relationship. The spermine-induced increase i n rectification on depolarization would reduce firing threshold. This provided rationale to determine i f the spermine-induced reduction o f the 2+  action potential threshold also depended on extra-and intracellular [Ca ]. The omission 2+  of C a  2+  from the A C S F , which normally contained 2 m M C a , decreased the threshold  from -53.7 ± 2.9 m V to -60.6 ± 3.1 m V (n = 3, A N O V A , P < 0.05). O n application o f 2+  spermine i n A C S F that was nominally C a - free, there was no change i n the threshold (control, -60.6 ± 3 . 1 m V , and spermine, -60.2 ± 3 . 2 m V ; n = 3). Recovery o f the 2+  threshold from the effects o f the C a - free solution occurred at 10 m i n after returning to  Chapter 3. Results I Ran  -79-  Internal EGTA  Spermine Control  0 Ca *, Spermine.Washout + 0 Ca * 2  J  2  Current step amplitude (pA)  B Internal BAPTA Spermine  Current pulse amplitude (pA)  2+  Figure. 3.13. Alterations in extra- and intracellular C a influence spermine effects on depolarizing current - voltage (V - I) relationships i n M G B neurons. ( A ) Voltage responses to current pulses (80 p A , 500 ms i n upper traces) and V -1 diagram show that 2+  removal o f extracellular C a  from A C S F abolished the increase i n voltage responses 2+  induced by spermine during internal application o f E G T A (10 m M ) . Perfusion o f C a 2+  free media for 6 m i n (0 C a ), alone, and with spermine (100 | i M , 3 min) did not alter voltage response. After a 10 m i n washout, and perfusion with control media containing 2 2+  m M C a (10 min), spermine application (100 | i M , 3 min) increased the voltage response. V -1 diagram i n same neuron shows that spermine did not change the slope o f the voltage 2+  responses during C a - free perfusion. When applied during extracellular perfusion with 2+  normal [Ca ], spermine increased the voltage responses to current pulses that were > 25 p A . (B) Voltage responses to current pulses (80 p A , 500 ms i n upper traces) and V -1 diagram show that spermine did not increase the voltage responses i n a neuron recorded during internal B A P T A (10 m M ) . Vertical bar, 10 m V ; horizontal bar, 100 ms.  Chapter 3. Results I Ran  -80-  2+  normal C a  perfusion. A subsequent application o f spermine for 3 m i n i n normal 2+  solution (2 m M C a ) reversibly reduced the firing threshold to -60.0 ± 2.6 m V (n = 3, A N O V A , P < 0.05). These effects, observed when the pipette solution contained 10 m M E G T A , were largely reversible (recovery, -55.3 ± 2.3 m V ) . The spermine-induced 2+  reduction i n action potential threshold was re-examined using the fast C a BAPTA  chelator,  (10 m M ) , applied internally. A 3 m i n application o f spermine did not  significantly change the threshold i n four neurons recorded with B A P T A - c o n t a i n i n g pipettes (control, -49.3 ±2.1 m V , and spermine, -49.3 ± 2.3 m V ) . These experiments 2+  demonstrated that the effects o f spermine on action potential threshold depended on C a entry.  3.2.1.5. Low threshold Ca spike (LTS) firing 2+  2+  Application o f C a - free A C S F abolished the transient, low threshold spike (LTS), evoked at the offset o f hyperpolarizing current pulses or on step depolarization i n neurons 2+  held at hyperpolarized potentials. This blockade confirmed the C a  mediation o f the L T S  (Tennigkeit et al., 1996). Spermine application increased action potential firing on top o f a L T S i n only 10 out o f 19 neurons, in contrast to the increased tonic firing rate on spermine application, observed i n all neurons. A s shown i n Figure 3.14A, spermine induced an action potential on the rebound depolarizing response at the termination o f hyperpolarizing current pulses. In 5 out o f the 10 neurons hyperpolarized with D C to -80 m V , spermine application induced one or two action potentials on a subthreshold rebound response to hyperpolarizing current pulses. Spermine application increased the amplitude  Chapter 3. Results I Ran  -81-  o f the rebound L T S that did not reach action potential threshold i n the remaining 5 neurons. The effects o f spermine were reversible, requiring 20 to 40 m i n for recovery.  Blockade o f voltage-dependent N a channels with T T X did not significantly alter the +  ability o f spermine to enhance the L T S i n 6 neurons (cf. Figure 3.14A and B ) . During T T X blockade, the spermine enhancement o f the L T S depended on the holding potential. In these experiments, the L T S was evoked by injecting hyperpolarizing currents o f different amplitude into neurons held at different membrane potentials (Figure 3.14C). Co-application o f spermine (100 u M ) and T T X induced an L T S i n neurons at potentials that caused marked inactivation o f the L T S . A t potentials where an L T S was present, a spermine application increased its amplitude and rate o f rise (dV/dt). There was a greater increase i n the dV/dt o f the L T S when the neuron was held at -55 m V than at -85 m V (Figure 3.14C). After spermine application, the L T S evoked in a neuron held at - 55 m V had an average dV/dt o f 3.1 ± 0 . 2 mV/ms, compared to 1.5 ± 0.3 m V / m s i n the control during T T X application. The average rate o f decay was - 1.7 ± 0.3 m V / m s (n = 6) with fast (26 ± 5 ms, n = 5) and slow (146 ± 12 ms, n =5) components (trace 2, Figure 3.14B). Figure 3.14C summarizes the effects o f spermine on the dV/dt o f the L T S , showing a maximal effect at a holding potential ( V ) = - 55 m V and a minimal increase at Vh = -85 n  m V i n 6 neurons (paired Mest, P < 0.01).  Since the hyperpolarization-activated current influences the rate o f rise o f the L T S , the next experiments examined whether spermine affected the voltage sag, mediated b y this current (Tennigkeit et al., 1996). The voltage sag was not prominent i n the majority o f  Chapter 3. Results I Ran  - 82 -  neurons. Spermine application (100 p M ) produced no change in the voltage sag induced by a hyperpolarizing current pulse i n three neurons. Hence, the increase i n the rate o f rise o f the L T S did not likely involve interactions with this hyperpolarization-activated conductance.  Due  to the previous observations, it was necessary to establish i f the  spermine  potentiation o f the L T S involved interactions at N M D A receptors (n = 8). In six o f these neurons, A P V application decreased the amplitude o f the subthreshold responses to current pulses (cf. Figure 3.14D, lower traces). A s shown for the neuron o f Figure 3.14D, A P V application also decreased an L T S burst to a single action potential and L T S rate o f rise, increasing the latency to the first action potential on top o f the L T S . Despite A P V antagonism o f N M D A receptors, spermine application transformed the subthreshold response into an L T S , as well as decreased the latency to an action potential on top o f the L T S (cf. A P V and spermine traces i n Figure 3.14D). In eight out o f eight neurons, A P V application (50 p M , 6 min) reduced the average rate o f rise o f the L T S from 1.6 ± 0.3 m V / ms i n naive controls to 1.2 ± 0.2 mV/ms. A subsequent co-application o f A P V and spermine caused a significant increase i n the rate o f rise o f the L T S to 2.2 ± 0 . 1 m V / m s ( A N O V A , P < 0.05).  In light o f the previous observations, it was necessary to determine i f the spermine potentiation o f the L T S involved interactions with A M P A receptors. During blockade o f A M P A receptors with C N Q X (30 p M , 6 min), spermine significantly increased the dV/dt of the L T S (control, 1.8 ± 0.1 mV/ms and spermine, 3.6 ± 0.2 m V / m s ; n = 5, A N O V A , P  Chapter 3. Results I Ran  -83 -  A  Control  J  Spermine + APV  APV  1  I  I  I  Washout (in APV)  I  _f  Figure 3.14. Effects o f spermine (100 p M , 3 min) on the low threshold C a spike ( L T S ) firing. ( A ) Superimposed voltage responses (control, spermine, and recovery) show that spermine induced an L T S on termination o f a hyperpolarizing current pulse ( - 40 p A , 500 ms). (B) Spermine increased the rate o f rise and amplitude o f the L T S at the end o f a hyperpolarizing current pulse ( - 80 p A ) just before (1), during (2), and after (3) spermine application during T T X blockade (0.6 p M ) o f voltage-dependent N a conductances. Holding potential, - 55 m V . (C) Bar graph summarizes spermine effects on dV/dt o f the L T S at the end o f hyperpolarizing responses and during T T X blockade. Spermine increased dV/dt i n neurons held at - 85, - 75, - 65, and, maximally, at - 55 m V (n = 6, paired t-test, *P < 0.01, **P < 0.005). (D) A P V (50 p M ) did not block the effects o f spermine on the L T S , as shown by sub-and suprathreshold responses to current pulses (60, 120 p A ) during application o f A P V , alone, and co-application with spermine. Application o f A P V reduced the subthreshold response, L T S rate o f rise, and number o f action potentials. Co-application (3 min) o f A P V and spermine transformed a subthreshold response to an L T S , increased L T S rate o f rise, and shortened the latency to the action potential. Recovery (in A P V ) was observed after a 10 m i n washout. Vertical bar, 15 m V i n ( A ) and (B) ; 30 m V i n (D). Horizontal bar, 150 ms. Insert i n (B) (right): Vertical bar, 3 m V ; horizontal bar, 30 ms. +  Chapter 3. Results I Ran  -84-  < 0.05). Hence, the effects o f spermine on the L T S did not likely involve N M D A or A M P A receptor interactions.  3.2.1.6. Excitatory and inhibitory postsynaptic potentials Spermine application (100 | i M ) to 18 neurons resulted i n bursts o f action potentials on E P S P s evoked by electrical stimulation o f corticothalamic projections (Figure 3.15A). Spermine had little or no effects on the rate o f rise o f the E P S P , but always prolonged the decay phase. The E P S P amplitude increased slightly ( 3 - 5  m V ) during spermine  application to five neurons, but this was not a consistent finding i n the 18 neurons. The spermine-induced  action potentials on the EPSPs were reversible i n all neurons.  Complete recovery was observed i n 13 o f 18 neurons at 35 m i n after terminating the application.  Spermine prolonged the E P S P decay time constant  (idecay),  as estimated with an a -  function fit o f the EPSPs (Figure 3.15B). This promoted the occurrence o f action potentials on top o f the EPSPs (Figure 3.15A). The E D o for the spermine-induced 5  increase i n  T  ueC  ay  o f EPSPs was - 1 0 0 pJVI which was approximately the same for the  spermine-induced increase i n firing (cf. Figure 3.9B). Recovery to the control value occurred after 30 m i n (148 ± 1 5 ms). Figure 3.15B summarizes these results for 15 neurons. Table 3.1 summarizes the effects o f single or cumulative applications o f spermine on the 90 - 10% decay time i n 18 neurons. Spermine application (100 u M ) did not significantly affect the amplitude or time course o f depolarizing potentials evoked by  Chapter 3. Results I Ran  -85-  Figure 3.15. Spermine (100 u M , 3 min) prolonged late component o f corticothalamic E P S P s mediated by N M D A receptors. ( A ) Spermine increased E P S P amplitude and duration, resulting i n three action potentials. (B) Spermine delayed the late component (2) o f the EPSPs. The bar graph summarizes the spermine-induced prolongation o f E P S P decay time constant (Xtay), expressed as % o f the control. Control x ^ w a s 142 ± 8.5 ms (n = 15, paired t-test, *P < 0.01). (C) Spermine did not affect E P S P s during N M D A receptor blockade by 50 uJVl A P V or significantly change remaining E P S P components. Bar graph summarizes the reduction i n E P S P Xtay by A P V and lack o f spermine effect during A P V blockade, expressed as % o f control. Control Tdecay W3.S 143.6 ± 14 ms (n = 8, A N O V A , *P < 0.01). Vertical bar, 5 m V ; horizontal bar, 200 ms.  Chapter 3. Results 1 Ran  Table 3.1: Effects o f spermine on E P S P variables  Amplitude (mV)  Rise (ms)  Decay (ms)  Half-width (ms)  n  Control Spermine  6.3 ± 1 . 8 9.1 ± 2 . 3  37 ± 6 2 0 ± 11  477 ± 11 710 ± 2 3 *  238 ± 1 1 297 ± 85  19 19  APV APV + Spermine  6.4 ± 2.1 6.1 ± 1 . 9  1 2 ± 1.1 12 ± 1 . 6  147 ± 9 . 5 153 ± 12  59 ± 4.2 63 ± 2.4  9 9  CNQX CNQX + Spermine  6.2 ± 0.8 6.3 ± 0 . 9  90 ± 5 95 ± 4  534 ± 1 8  247 ± 9.5 433 ± 20**  9 9  Arcaine Arcaine + Spermine  4.5 ± 1 . 4 4.6 ± 0 . 6  36 ± 7 42 ± 1 1  419 ± 11 394 ± 15  204 ± 40 205 ± 38  5 3  Glycine Glycine + Spermine  6.9 ± 1 . 1 9.0 ± 2.8  24 ± 5 28 ± 9  190 ± 7 400 ± 2 1 *  245 ± 41 437 ± 4 5  3 3  Values are m e a n ±  SE.*P<  **  925 ± 24  0.05 , ** P<  0.01, Mest  Chapter 3. Results I Ran  - 87 -  stimulation o f the brachium colliculi inferioris (n = 6 ; data not shown). These potentials, 100 to 200 ms i n duration, were likely IPSPs mediated by G A B A A receptors because they reversed at potentials near Eci (-55 m V ) and were sensitive to blockade by bicuculline (50 LiM, n = 6).  Spermine prolonged the decay o f the late E P S P component mediated by an N M D A - t y p e receptor. The application o f A P V (50 nJVl), blocked the late component, resulting i n shorter rise (10 - 90%) and decay (90 - 10%) times o f the EPSPs (Table 3.2). During N M D A receptor blockade, E P S P mean idecay was 100 ± 14 ms, compared to 143 ± 15 ms in control (Figure 3.15C; n = 8, A N O V A , P < 0.01). This mean value did not change significantly during combined spermine and A P V application (Figure 3.15B).  Interactions with A M P A receptors, which might prolong the EPSPs, were examined by applying spermine i n combination with C N Q X (30 | i M , 6 min). Spermine prolonged the E P S P 90 - 10% decay time during C N Q X blockade to the same extent as i n the absence of A M P A receptor blockade (Table 3.1). In 3 additional neurons, spermine was applied before the co-application with C N Q X . The co-application resulted i n a significant prolongation o f the E P S P to the same extent as in the absence o f C N Q X blockade o f A M P A receptors (Table 3.1). This confirmed that spermine affected only the N M D A mediated component. Co-application o f C N Q X  (30 | J M ) and A P V (50 L I M ) then  abolished the early and late components o f the E P S P which remained absent despite a subsequent spermine application (n = 4). These results suggest that spermine increased the duration o f the E P S P decay phase by interacting with N M D A receptors.  Chapter 3. Results I Ran  - 88 -  The possibility was considered that spermine prolonged the E P S P s by acting on an extracellular polyamine-sensitive site o f the N M D A receptor (cf. Benveniste and Mayer, 1993). This required an investigation o f the interactions o f spermine and arcaine, an antagonist that acts at the polyamine-sensitive site on the N M D A receptor (Reynolds, 1990). In these studies, arcaine (40 p M ) , spermine (100 p M ) , arcaine and spermine, were sequentially applied each for 3 m i n (n = 3). Arcaine, alone, did not greatly alter the configuration o f the E P S P (Figure 3.16A) or produce changes i n the E P S P amplitude, 90 - 10% decay time, and half-width (Table 3.1). After a 15 m i n washout from arcaine application, spermine significantly prolonged the E P S P  Xdecay  to 180 ± 3 2 ms from 101 ±  16 ms i n the control (Figure 3.16A). A subsequent co-application o f spermine and arcaine abolished the actions o f spermine, resulting i n  Tdecay  o f 118 ± 16 ms (Figure 3.16A). The  graph o f Figure 3.16A summarizes the spermine-induced increases i n E P S P  Tdecay  and  arcaine blockade o f spermine effects.  A possibility was tested that spermine increased the N M D A - m e d i a t e d component o f the E P S P by potentiating the actions o f glycine on the N M D A receptor. In the presence o f a saturating concentration o f glycine (40 p M ) , spermine still prolonged the E P S P by ~ 4 9 % (Figure 3.16B). In 3 neurons, spermine increased E P S P  Tdecay  from 255 ± 44 ms to 379 ±  53 ms ( A N O V A , P < 0.05). In summary, spermine actions on the E P S P likely involved an extracellular polyamine-sensitive site, and not a glycine-sensitive site o f the N M D A receptor.  Chapter 3. Results I Ran  Control  Arcaine  Control  -89-  Spermine  Spermine + Arcaine  Spermine Spermine + Arcaine  Control (Glycine)  Spermine + Glycine  Control  Washout (Glycine)  Spermine Washout  Figure 3.16. Spermine (100 U . M ) prolonged the E P S P s by interacting with the polyaminesensitive site on N M D A receptor. (A) Arcaine (40 | i M ) , a blocker at the polyaminesensitive site on N M D A receptors, almost eliminated the spermine-induced prolongation o f the E P S P , expressed as % control Xdecay which was 100.4 ± 16 ms (n = 3, A N O V A , *P < 0.05). Note that there was a 10 min washout after arcaine, prior to spermine application. (B) Spermine (100 u M ) prolonged the E P S P Xdecay during co-application with glycine (10 JIM). Control Xdecay (glycine present) was 255 ± 44 ms (n = 3, paired Mest, *P < 0.05). Bar graphs summarize the effects o f spermine on E P S P Xdecay during arcaine and glycine applications. Holding potential, - 60 m V . Vertical bar, 5 m V ; horizontal bar, 100 ms.  Chapter 3. Results 1 Ran  -902+  2+  A contribution o f extracellular C a  or M g  to the spermine-induced enhancement o f  E P S P s was assessed i n the next experiments. Spermine application did not alter the am2+  plitude or duration o f the EPSPs during a 6 m i n perfusion o f C a - free A C S F i n three neurons (data not shown). Hence, spermine effects on N M D A - m e d i a t e d E P S P s likely 2+  depended on C a  entry.  2+  In 2 neurons, the omission o f M g  from A C S F perfusion resulted i n subthreshold  oscillations o f the membrane potential and spontaneous firing o f action potentials. These observations were consistent with previous studies on thalamocortical neurons (Jacobsen 2+  et al., 2001) which prevented critical assessment o f a co-agonist role o f M g  at the  polyamine-sensitive site on the N R 2 B receptor subunit (Kew and Kemp, 1998).  3.2.2. Pentobarbital effects on corticothalamic EPSPs The  following  experiments  were  performed  in  thalamic  neurons  of  the  ventroposteriolateral thalamic nucleus o f the rat (see Methods).  In non-thalamic neurons, pentobarbital, at anesthetic doses, inhibits N M D A - m e d i a t e d currents (Charlesworth et al., 1995). This provided some rationale to test whether pentobarbital could depress evoked corticothalamic responses mediated by  NMDA  receptors i n V B neurons. Pentobarbital at 200 p M , but not at 50 p M shortened the duration o f the N M D A - m e d i a t e d EPSPs (Figure 3.17A). This result was consistent with pentobarbital's shortening o f N M D A receptor mean open time observed i n hippocampal neurons (Charlesworth et al., 1995).  Chapter 3. Results I Ran  -91-  The depressant effect o f pentobarbital suggested that it would reduce E P S P prolongation caused by spermine. A n application o f spermine, at 100 p M , resulted i n ~ 4 5 % prolongation o f EPSPs (Figure 3.17B). This observation was consistent with E P S P prolongation i n medial geniculate neurons o f gerbils (cf. Figure 3.15). During spermine application, a subsequent co-application o f pentobarbital at 50 p M did not produce a significant change i n the duration or amplitude o f N M D A - m e d i a t e d E P S P s . However, a subsequent increase i n the pentobarbital dose to 200 p M produced a reversal o f the spermine-mediated prolongation o f the E P S P (Figure 3.17). Pentobarbital reversal o f the prolongation o f EPSPs caused by spermine implicated an action at specific modulatory sites.  3.2.3. Interactions ofZn  2+  with spermine and pentobarbital  The first possibility examined was that pentobarbital reversal o f spermine prolongation o f E P S P s involved interactions at the Z n  2 +  binding site on N M D A receptors. This was done  by applying Z n , a negative modulator o f N M D A receptors at a site distinct from that o f 2 +  polyamines (Forsythe et al., 1988). Application o f Z n  2 +  (20 p M , 1 min) resulted i n a 32 %  reduction i n E P S P decay time constant (Figure 3.17C). Z n  2 +  application also reduced  E P S P mean amplitude, however, this was not significant (6.1 ± 1 m V in controls compared to 4.9 ± 0.9 m V after Z n , n = 5, P > 0.05). During Z n 2 +  2 +  application, co-  applied spermine (100 p M , 1 min) prolonged the E P S P decay by 58 % (137 ± 12 ms). A subsequent co-application o f pentobarbital reversed the spermine-mediated prolongation of E P S P to control levels (with Z n  2 +  present, Figure 3.17), similar to the effects i n the  Chapter 3. Results  -92-  1 Ran  Figure 3.17. Pentobarbital effects on NMDA-mediated corticothalamic EPSPs. ( A ) Pentobarbital at 200 f i M , but not at 50 p M shortened the duration o f the N M D A mediated EPSPs. The bar graph summarizes the effects on E P S P decay time constant (Tdoay), expressed as % o f the control. Control T ^ y was 118 ± 13 ms (n = 6, A N O V A test, *P < 0.05- significantly different from control). (B) Pentobarbital at 200 p M , but not at 50 p M reversed the spermine-mediated (100 p M , 1 min) prolongation o f the N M D A mediated EPSPs. The bar graph summarizes the pentobarbital-reversal o f spermine prolongation o f E P S P decay time constant (tdecay), expressed as % o f the control. Control Tdecay was 123 ± 11 ms (n = 5, A N O V A - t e s t , *P < 0.05 - significantly different from spermine 100 p M + pentobarbital 0 p M ) ) . (C) Z n (20 p M , 1 min) reduced the amplitude and shortened the duration o f NMDA-mediated EPSPs. Graph summarizes the effects on Tdecay as i n A (n = 5, student Mest, *P < 0.05- significantly different from control). (D) The presence o f Z n did not alter spermine's ability to prolong the N M D A mediated E P S P . A subsequent co-application with pentobarbital still reversed the E P S P prolongation caused by spermine. Graph summarizes the effects on prolongation and its reversal by pentobarbital. Tdecay as i n A (n = 5, A N O V A - t e s t , *P < 0.05- significantly different from control or spermine + pentobarbital). EPSPs were evoked by internal capsule stimulation (50 V , lOOps). Holding potential, - 60 m V . C N Q X (30 p M ) was applied throughout the experiment. E P S P traces are averages o f 5 samples each. 2 +  2 +  Chapter 3. Results I Ran  - 93 -  absence o f Z n .  These observations suggested that pentobarbital acted at sites distinct  from those o f Z n  , possibly the polyamine site.  2 +  3.2.4. Antagonism of polyamine site The lack o f effect o f Z n  2 +  on pentobarbital reversal o f spermine prolongation o f E P S P s  implicated interactions at the polyamine site. To test this hypothesis, spermine and pentobarbital were co-applied during blockade o f the polyamine site with arcaine. B y itself, arcaine (40 L I M , 1 min) decreased the duration o f EPSPs to 63 % o f the control (Figure 3.18); an effect which indicated that endogenously-released spermine contributed to E P S P decay. During arcaine application, a subsequent co-application o f spermine did not alter the amplitude or duration o f EPSPs (Figure 3.18). A subsequent co-application with pentobarbital (200 m M , 3 min) did not change E P S P duration. The results suggested that the reversal o f spermine prolongation o f EPSPs involved an allosteric modulatory action o f pentobarbital at the polyamine site on N M D A receptors.  3.2.5. Discussion  Spermine application increased the decay time constant o f corticothalamic E P S P s mediated by N M D A receptors. This finding is consistent with the increased amplitude o f N M D A - e v o k e d currents during spermine application to cultured hippocampal and spinal neurons (Lerma, 1992; Benveniste and Mayer, 1993). In the present studies, the actions of spermine were selective and required extracellular C a  2 +  because they were abolished i n  three neurons by brief Ca -free perfusion. Spermine also did not significantly alter the 2+  Chapter 3. Results I Ran  -94-  A  Figure 3.18. Pentobarbital reversal o f spermine E P S P prolongation involves interactions at the polyamine site on N M D A receptor. A ) Application o f arcaine (40 | i M , 1 min) shortened the duration o f N M D A - m e d i a t e d EPSPs without affecting their amplitude. The bar graph summarizes s the effects on Xdecay i n five neuorns. B ) W i t h arcaine present, spermine (100 u\M, 3 min), when applied alone, or in combination with pentobarbital (200 | i M , 3min) did not cause any prolongation o f the E P S P . Holding potential, - 60 m V . The bar graph summarizes this lack o f effect (n = 5, P > 0.05 with or without spermine). E P S P traces i n A and B are averages o f 5 samples each.  Chapter 3. Results I Ran  -95-  early E P S P component mediated by A M P A receptors, or appreciably affect IPSPs mediated by G A B A receptors that were sensitive to bicuculline antagonism. Application of A P V completely blocked the spermine-induced increase i n the E P S P decay time constant. This implicated N M D A receptors in spermine actions to increase excitation.  The effects o f spermine on M G B neurons involved a polyamine-sensitive site on the NR2B  subtype o f N M D A  receptors,  as demonstrated  with  arcaine and  glycine  applications. Arcaine, itself, did not have significant effects on the passive and active membrane properties but reversed the spermine-induced decrease o f the E P S P decay. Previous studies have shown that arcaine blocks spermine actions by inverse agonism, antagonism, and open-channel blockade o f the polyamine-sensitive site on N M D A receptors (Reynolds, 1990; Pritchard et al., 1994). The actions o f spermine at this site decreased  the  EPSP  decay, despite  saturating  concentrations  o f glycine.  These  observations are consistent with the glycine-independent potentiation o f N M D A currents by spermine at the N R 2 B receptor subunit i n cultured hippocampal neurons (Benveniste and  Mayer, 1993). In thalamocortical neurons, the persistence during high glycine  concentrations and arcaine reversal imply that spermine acted independently o f the glycine site at a specific polyamine-sensitive site on the N R 2 B receptor subunit o f the N M D A receptor.  Chapter 3. Results (Ran  -96-  The N R 2 B subunit may modulate the decay time constant o f the N M D A  receptor-  mediated E P S P during the development i n M G B neurons. A t the end o f the second postnatal week, thalamocortical neurons express an abundance o f the N R 2 B polyaminesensitive receptor subtype i n the M G B and lateral geniculate body ( L G B ) o f the rat (Chen and Regehr, 2000). The duration o f EPSPs mediated by N M D A receptors i n L G B neurons o f the rat is similar at P14 to that i n gerbil M G B neurons. The decay time constant i n L G B neurons is longer at P14 i n rats than at earlier (P7-P13) or later (PI 6 P28)  stages o f development (Chen and Regehr, 2000; cf. also rat M G B at P 2 1 - P 4 2 ,  Bartlett and Smith, 1999). Hence, spermine modulation o f the N R 2 B subunit may cause the longer E P S P duration i n M G B neurons at P I 4 .  Spermine enhanced excitability by increasing inward rectification on depolarization, without greatly affecting the passive properties o f M G B neurons. It is not known i f the passive and active membrane properties o f M G B neurons mature by P14 i n gerbil, as i n the rat (Tennigkeit et al., 1998). Thalamocortical neurons o f the adult guinea p i g and P 7 P28  rat inwardly rectify because the activation o f persistent N a  +  conductance on  depolarization results i n an amplification o f the voltage response (Jahnsen and Llinas, 1984; Tennigkeit et al., 1998; Parri and Crunelli, 1998). In the present studies, blockade of the TTX-sensitive rectification or N M D A receptors eliminated the spermine-induced enhancement o f rectification on depolarization. These findings imply that spermine interactions with N M D A receptors led to activation o f a persistent N a conductance i n +  M G B neurons.  Chapter 3. Results I Ran  - 97 9+  An  elevation i n intracellular C a  9+  concentration  ([Ca ];) due to N M D A  receptor  activation (Jahr, 1992) may have enhanced rectification on depolarization. In the present study, there was no evidence for a spermine-induced increase i n voltage responses on depolarization during Ca -free perfusion or rapid chelation o f C a 2+  2 +  with intracellular  B A P T A . It seems likely that an elevation o f [Ca ]j initiated by spermine actions at 2+  NMDA  receptors activated  intracellular messengers and increased  this  rectifying  behavior. In neocortical neurons, transmitter activation o f dendritic N M D A receptors increases  Ca  2 +  entry  (Schwindt  and  Crill,  1995)  phosphorylation (Siekevitz, 1991) and a persistent N a  that +  may  increase  channel  conductance (Schwindt et al.,  1992). Hence, the spermine-induced enhancement o f TTX-sensitive rectification on depolarization may result from N M D A - m e d i a t e d C a  2 +  entry i n M G B neurons.  The effects o f spermine on membrane rectification and firing threshold may involve the recruitment o f a Ca -dependent 2+  second messenger, subsequent to N M D A  activation. Activation o f N M D A receptors enhances C a  2 +  receptor  entry, resulting i n a C a  2 +  gradient i n the dendrites (Connor et al., 1988) and activation o f a protein kinase C ( P K C ) pathway. A rise i n intracellular [Ca ] also may activate calmodulin kinase II which 2+  enhances N a  +  currents (Carlier et al., 2000). P K C activation increases membrane  excitability by shifting the activation curve for the persistent N a  +  current along the  voltage axis to more hyperpolarized potentials (Astman et al., 1998; Franceschetti et al., 2000). This voltage range is consistent with the range o f spermine enhancement o f voltage rectification i n our experiments.  Chapter 3. Results I Ran  -98-  The increased rectification on depolarization may have reduced the threshold for an action potential i n M G B neurons (cf. neocortical neurons, Stafstrom et al., 1982). Antagonism o f N M D A receptors, perfusion with Ca -free A C S F or rapid chelation o f 2+  Ca  with B A P T A , eliminated the reduction i n threshold and increased tonic firing due to  spermine application. Hence, the modulation o f N M D A receptor-mediated C a  2 +  entry  likely increased membrane rectification on depolarization and reduced firing threshold. This mechanism explains the ability o f spermine to increase postsynaptic excitability and tonic firing i n M G B neurons.  Spermine facilitated L T S firing by a mechanism that did not involve interactions with N M D A receptors. Spermine increased the rate o f rise and amplitude o f the L T S , despite A P V blockade o f N M D A receptors. This was evident on depolarization to action potential threshold where there is a smaller gradient for C a inactivation o f T-type C a  2 +  2 +  as well as greater  channels (Hernandez-Cruz and Pape, 1989). Spermine  enhanced the L T S during blockade o f voltage-dependent N a channels by T T X . Hence, a +  change i n some parameter o f the T-type C a  2 +  current, e.g. voltage dependence o f the  inactivation-activation relationship, may have increased the L T S .  Pentobarbital modulated N M D A - m e d i a t e d  corticothalamic EPSPs producing  more  transient responses. A t an anaesthetic concentration, pentobarbital shortened the duration of  NMDA-mediated  corticothalamic  EPSPs.  Pentobarbital  reversed  spermine  Chapter 3. Results I Ran  -99-  prolongation o f corticothalamic EPSPs by a mechanism that was independent o f Z n interactions. The lack o f pentobarbital effects during arcaine blockade implicated an action at the polyamine site on N M D A receptors. These effects are consistent with pentobarbital shortening o f burst durations o f N M D A - m e d i a t e d single channel currents (Charlesworth et al., 1995). Pentobarbital actions on N M D A - m e d i a t e d corticothalamic transmission may contribute to its anti-epileptic effects.  The depressant effects o f pentobarbital on N M D A - m e d i a t e d synaptic responses provided a rationale to examine its short-term effects on pre- and postsynaptic parameters o f nonN M D A mediated transmission presented i n the next section.  Part III. Effects of pentobarbital on short-term depression 3.3.1. Behaviour of EPSCs in trains during short-term depression 3.3.1.1 Passive membrane properties For proper assessment o f short-term depression (STD), Q X - 3 1 4 and Cs-gluconate were applied  intracellularly to  block, respectively, N a  +  and  K -channels and +  reduce  postsynaptic currents. W i t h this pipette solution, the input resistance (Rj) increased b y ~ 81 % (380 ± 25 MQ., n = 10, P < 0.05) compared to values obtained using solutions containing K -gluconate and no Q X - 3 1 4 (210 ± 15 MQ, n = 9). During combined C s +  +  and Q X - 3 1 4 blockade, pentobarbital (200 p M ) did not alter the R i throughout 3 - 5 m i n of application (365 ± 34 M Q , n = 10; cf. W a n et al. 2004). Hence, intracellular blockade  Chapter 3. Results I Ran  of N a  +  and K  +  -100 -  channels reduced their postsynaptic contributions to stimulus-evoked  depression (Cahalan and Aimers 1979; Konishi 1990; Budde et al. 1994).  The experiments were performed in neurons voltage clamped at -80 m V i n order to minimize  postsynaptic  contributions  of  voltage-dependent  (Hernandez-Cruz and Pape, 1989). This allowed the study o f the  Ca  2 +  conductances  frequency-dependent  aspect o f corticothalamic S T D while minimizing postsynaptic temporal summation.  3.3.1.2. Frequency - dependent fade (STD) of corticothalamic EPSCs Repetitive stimulation i n the 2.5 - 20 H z range produced S T D o f E P S C s (Figure 3.19). W i t h increasing stimulation frequencies, the train o f E P S C s decreased i n amplitude to a plateau (Si . o) that was 49 to 21 % o f the 1 E P S C amplitude ( S i ; see Plateau/Si ratio i n st  5  2  Table 3.2.1 A ) . The relation between the plateau and stimulation frequency is illustrated i n their product  value (plateau  x H z ) which increased  significantly at stimulation  frequencies > 5 H z (Table 3.2.1 A ) . The apparent Q N decreased at stimulation frequencies > 10 H z , indicating substantial refill at lower frequencies (Table 3.2.1 A ) . The ratio o f the amplitude o f the 12 amplitude  (S12/S10),  th  E P S C , subsequent to the omitted 1 1 increased at stimulation frequencies  th  stimulus to the 10  th  EPSC  > 5 H z (Table 3.2.1 A ) ,  indicating high values o f fractional release i n the plateau. The  frequency-dependent  characteristic o f S T D was similar to other observations at corticothalamic synapses ( L i et al. 2003; Reichova and Sherman, 2004).  Chapter 3. Results I Ran  - 101 -  2.5 Hz  A  10 Hz  5 Hz  20 Hz  ({{fmfrrrrcrrm—  B  1 nA 1.00  Ui% |  Z  |  0.7S 0 C  ^  °'  x  I f  0.25 ooo-  10  15  »>  !0  15  B>  stimulus #  sllmulus #  Figure 3.19. Frequency-dependence o f corticothalamic S T D . A ) Traces o f trains o f E P S C s during S T D . Increasing the frequency o f stimulation from 2.5 to 20 H z enhanced S T D . Expanded traces below show the 1 to 5 E P S C s (middle) and the 1 0 to 1 4 E P S C s around the omitted 1 1 stimulus (bottom). B ) Normalized E P S C amplitudes at four stimulation frequencies. A t 10 H z , E P S C s reached a plateau o f 40 % o f the 1 response whereas at 20 H z E P S C amplitudes reached a plateau o f 25 % o f the 1 response. A t 20 H z stimulation, the mean amplitude o f the 1 1 response just subsequent to the missing 1 0 stimulus nearly doubled, consistent with the depletion model. Traces in A are averages o f 6 repeats from 1 neuron. Values i n B are averages o f 6 neurons. S E M indicates between neuron variations. Neurons were held at - 80 m V . st  t h  th  th  th  st  st  th  th  Chapter 3. Results I Ran  Table 3.2.1 A : Summary o f parameters o f corticothalamic S T D at different frequencies Frequency (Hz)  2.5  5  10  20  Parameter S,(nA)  1.19 ± 0 . 3 5  1.21 ± 0 . 3 8  1.18 ± 0 . 2 9  1.17 ± 0 . 4 1  S (nA)  0.67 ± 0.23  0.70 ± 0.25  0.74 ± 0 . 1 9  0.56 ± 0.32  S2/S1  0.56 ± 0 . 1 9  0.57 ± 0.2  0.63 ± 0 . 1 6  0.47 ± 0.27  S12/S10  1.02 ± 0 . 0 8  1.06 ± 0 . 0 9  1.31 ± 0 . 1 0 *  1.86 ± 0 . 1 1 *  Plateau  0.58 ± 0 . 1 6  0.52 ± 0 . 1 9  0.41 ± 0 . 1 7  0.25 ± 0 . 1 5  0.49 ± 0 . 1 3  0.43 ± 0 . 1 5  0.35 ± 0 . 1 3  0.21 ± 0 . 1 2 *  Plateau x H z (nA/s)  1.4 ± 0 . 4  2.6 ± 0 . 5  4.1 ± 0 . 6 *  5.0 ± 0 . 6 *  Apparent Q N (nA)  4.25 ± 0 . 1 1  4.22 ± 0 . 1 0  4.09 ± 0 . 1 3  3.26 ± 0 . 1 4 *  2  (Si 5-20) (nA) Plateau/Si  Values are mean ± S E M between neurons; n = 6, * (relative to 2.5 H z ) P < 0.01, A N O V A test  Chapter 3. Results I Ran  -103 -  S T D was found to be associated with negative covariances between pairs o f the first 5 E P S C s , consistent with the binomial/depletion model (Figure 3.20). Theoretically, i n the absence o f refill, the covariance divided by the product o f the mean amplitudes o f the 1 and 2  n d  st  E P S C s (<Si>-<Sj>) equals the negative o f the reciprocal o f the number o f release  sites (-1/N). The plot o f Figure 3.20A shows the negative o f the covariance, expressed as -cov(Si,Sj)/ <Si>-<Sj> between the 1 E P S C and the 2 st  n d  to 5 E P S C s . A t each particular th  frequency, the covariance term decreased as the distance between the pairs o f stimuli increased (Figure 3.20). This attenuation o f the covariance term is expected from refill which reduces the negativity o f the covariance term. A s shown i n Figure 3.20, an increase in the stimulation frequency worked i n the opposite direction, as expected from a lower a between stimuli. The covariance data were entirely consistent with the binomial/depletion model (see Methods). This validated the use o f the covariance term to correct the variance to mean ratios and to estimate the apparent quantal sizes during S T D , at each stimulus i n the train.  The results indicated an inconsistency with the simple model. Namely, S T D was also characterized by a frequency-dependent decrease i n the apparent quantal size along the E P S C train (Figure 3.20 B , Table 3.2.IB). After 2 stimuli, the apparent quantal size declined to a plateau value lower than the initial. The equivalence with variance/mean ratios indicated no error in the correction, which is intrinsically small when signals are much less than the initial (Figure 3.20A). The apparent quantal size values, i n the 26-35 p A range, were not significantly different from the mean amplitudes o f evoked miniature  Chapter 3. Results I Ran  - 104-  2.5 Hz  B  5 Hz  10 Hz  20 Hz 5"  0.04 0.03  i ° °^  2  10  .15  20  IS  1 1 0.02 I 0.01  0.02  20  Ifi.  20  aoo  Stimulus U  _ 50  SHi-Hii  10  15  : stimulus ft •  S 8  12  H t f H H  20  •e 40  •g »  10  15  20  stimulus #  10  15  20  stimulus #  Figure 3.20. Validation o f the corrected variance-mean method during corticothalamic S T D . A ) Negative covariances within the first five E P S C s during train-evoked S T D . Attenuation o f the covariance term calculated for pairs o f the 1 E P S C relative to the 2 to 5 E P S C s . The attenuation increased with decreasing stimulus frequencies i n the 2.520 H z range, likely due to refill o f depleted packets. B ) Quantal size estimates during S T D . Note the frequency-dependent reduction i n quantal size early i n the train and the lack o f change i n quantal size after an intra-train gap at the 1 1 stimulus. C) Alterations in quantal content during S T D at 2.5 - 20 H z . Note the similarity i n reduction o f quanta to the rundown i n E P S C amplitude (Figure 3.19) and the post intra-train gap increase i n quanta reflecting a presynaptic provenance. Data were obtained from same neurons as i n Figure 3.19. S E M indicates between neuron variations. The relative jump after the gap theoretically equals P utput (l-o0. st  th  th  x  O  n d  -105-  Chapter 3. Results I Ran  A  Pre-stimulation minEPSCs  B  2.5 Hz  (a)  1  Post-stimulation minEPSCs  s  50 pA  10 Hz  5Hz  20 Hz  Before trains 1-.0,  46 minis in 30s  _0.8  r  ^ - * - 11.4 ± 2.1 pA  . | 0.6  •  11 1 ±2.4pA  0.6. 0  0  10  20 30 Size (pA)  40  0  10  I !  |0.2.  a.  ?o:2  0.0  20 30 Size (pA)  40  53 minis in 30s  ,0.8] i  —  10.9 ±3.1 pA  ; BA  I'  9 OA 0.0  |  1.0,  48 minis in 30s  „0.B>  •1,1.8*2.5 f>A  f  1.0  43 minis in 30s  0.4  !"o,2.0  10  20 30 Size(pA)  40  0  10  20 30 Size (pA)  40  0.0 0  10  20: 30 Size (pA)  40  (b) After trains 44 minis in 6 s'  37 mins in 6 s  e  i  :1\ 0  10  20  30  40  0  10  Size(pA)  20 30, ,Size{pA)  40  (b)-(a) 3  ? I  31.613:0 pA —  I: 0  lifll  20 30 Size (pA)  3  20 30 Size (pA)  40  ±3, 40  I o -1  30.9 ± 2.4 p A : - » /  32.55 2 . 7 p A - « ^  I* | :  32,3±3:3pA»^  2  2  I o  10  6;  4  a  0  2  0  ' 1 *  20 30. Size (pA)  40  0 ^ior M^""30*''~*4o" =  Size(pA)  ! 0 •1-  '-1a  20  30  Size(pA)  Figure 3.21. Pre- and poststimulation miniature E P S C s i n a neuron vary i n size. A ) Sample records o f miniature E P S C s (minEPSCs), 5 s i n duration, before and after a 10 H z stimulus train. B ) (a) Amplitude histograms o f spontaneous m i n E P S C s counted 5 s before the stimulus train (6 repeats, 30 s in total), (b) Amplitude histograms o f evoked m i n E P S C s counted 1 s after the stimulus train (6 repeats, 6 s total). Total m i n E P S C count (minis) is indicated above the histograms. Evoked m i n E P S C sizes were obtained by subtraction o f spontaneous from evoked m i n E P S C histograms ((b) - (a)). Values next to black arrows are the mean size ± S E M . The apparent quantal sizes (Q') were (in p A ) : 34.6 at 2.5 H z , 33.1 at 5 H z , 32.9 at 10 H z , and 34.1 at 20 H z .  Chapter 3. Results I Ran  106-  Table 3.2.IB: Derived parameters o f S T D at different frequencies Frequency (Hz)  2.5  10  20  Parameter cov(Si,S ) (nA ) 2  0.016 ± 0 . 0 0 9  -0.018 ± 0 . 0 1 0  -0.021 ± 0 . 0 1 3  -0.024 ± 0 . 0 1 2  2  Q'(Si) (pA)  36.1 ± 9 . 1  37.3 ± 7 . 3  35.3 ± 6 . 2  36.5 ± 8.2  Q'(S ) (pA)  35.5 ± 8 . 7  34.9 ± 5 . 5  34.5 ± 6 . 4  36.1 ± 5 . 9  QXS15-S20)  31.3 ± 6.1  29.0 ± 7 . 3  26.1 ± 5 . 7  25.5 ± 5 . 4  2  (PA) QTSis^ai Q'(Si)  0.85 ± 0 . 0 7  0.78 ± 0 . 1 0  0.74 ± 0.08  0.68 ± 0.06  Var/Mean  30.7 ± 7.4  31.2 ± 6 . 6  27.2 ± 4 . 9  26.4 ± 5 . 6  (S15-S20)  (PA) Evoked m i n E P S C size (PA)  30.5 ± 3 . 7  31.4 ± 4 . 1  Pre-stimulation m i n E P S C size (PA)  11.4 ± 3.1  11.2 ± 2.9  32.0 ± 5 . 3  11.1 ± 3 . 4  29.1 ± 6 . 1  10.5 ± 2 . 5  mi  53 ± 4  52 ± 3  52 ± 8  53 ± 3  m 15-20  34 ± 3  31 ± 4  27 ± 3  21 ± 4 *  mu/mio  1.08 ± 0 . 1 1  1.13 ± 0 . 1 8  1.25 ± 0 . 2 0  1.96 ± 0 . 1 5  Values are mean ± S E M between neurons; n = 6 ; * (relative to 2.5 H z ) P < 0.05; data from same neurons as Table 3.2.1 A .  Chapter 3. Results I Ran  -107 -  E P S C s (minEPSC) observed 1 s after the stimulus train (Figure 3.21; Table 3.2.1B). The frequency o f these m i n E P S C s increased with stimulation frequency (Figure 3.21).  During S T D , the quantal content (m) also decreased to a plateau that depended on the stimulation frequency. A t 20 H z stimulation, m reached a plateau value (m 15.20) o f - 4 0 % o f the initial m (m\, Figure 3.20C). The m value increased subsequent to the gap at the omitted 1 1 stimulus (mn/mo', Figure 3.20C, Table 3.2.IB), consistent with the increased th  amplitude o f the 12  th  E P S C amplitude (20 H z i n Figure 3.19, Table 3.2.IB). This  observation was indicative o f a refill process that restores the apparent number o f releasable quanta. In summary, an intra-train reduction i n quantal content mediated much o f the frequency-dependent component o f S T D .  3.3.2. Effects of alterations in extracellular Ca concentration ([Ca ] ) 2+  2+  e  3.3.2.1 Low [Ca J perfusion 2+  e  Since C a  2 +  has been proposed to be a key factor for release probability and therefore a  mediator o f S T D , the following experiments examined i f S T D persisted under conditions o f low release probability. Washing extracellular C a  2 +  with E D T A resulted i n an overall  reduction i n the amplitude o f the E P S C train (Figure 3.22) and a rundown to a plateau o f 28 % o f the initial E P S C amplitude (Figure 3.22; Table 3.2.2A). A subsequent application o f a 0.2 m M C a  2 +  media with no E D T A increased the amplitude o f the E P S C train,  confirming an effective reduction o f extracellular C a  2 +  concentration ([Ca ] ) by the 2+  e  previous E D T A solution. Contrary to the expectation, there was a significant drop i n the apparent Q N i n low [ C a ] with or without E D T A , rather than a fall i n fractional release 2+  e  Chapter 3. Results I Ran  - 108 -  (Table 3.2.2A). In the neuron shown in Figure 3.22, an application o f D M S O (1%), subsequent to 0.2 m M [Ca ] , produced higher E P S C amplitudes and faster rundown by e  increasing the quantal contents at the beginning o f the train. Hence, a substantial decrease 2_|_  in [Ca ] did not result in a loss o f S T D . e  The reduction in Q N was, apparently, due to a decrease i n apparent quantal size throughout the train i n low [ C a ] , with or without E D T A (Figure 3.22; Table 3.2.2B). 2+  e  O n application o f very low [ C a ] (0.1 m M C a 2+  i n E D T A ) , the apparent quantal size  2 +  e  was decreased already at the 1 subsequent switch to 0.2 m M C a  st  response and in the remainder o f the train. The  2 +  (without E D T A ) increased the quantal content at the  beginning o f the train without having significant effect on quantal size (Table 3.2.2B). These effects o f low C a  2 +  on the quantal parameters suggested the participation o f high  probability release sites consistent with a compound binomial model (Brown et al. 1976; Walmsley et al., 1988). However, the high probability release sites were associated with relatively small quantal responses, perhaps at sites on dendrites relatively protected i n some way from reduction in local [ C a ] . 2+  3.3.2.2. Elevated [Ca ] perfusion e  A n increase i n [ C a ] 2+  enhanced S T D . Raising [ C a ] 2+  e  e  from 2 to 8 m M resulted i n  increased amplitudes o f the initial E P S C s (Figure 3.23). The E P S C s plateau increased by - 5 0 %, whereas, the ratio o f the plateau to the 1 E P S C (plateau/Si) decreased (Table st  3.2.2A). The ratio o f E P S C s around the intra-train gap ( S i / S i ) increased from 1.31 ± 2  0  0.08 to 1.56 ± 0.07 (n = 5, P < 0.05, Mest). Consistent with increases i n fractional  Chapter 3. Results I Ran  -109 -  Control  mm  EDTA  irfW  frr  0.2 mM Ca''  1% DMSO (0.2 mM Ca")  f/ffCfrfrrfrmrrr  mrrr  1 0,21 nAL 0.2s  nA  / f t «  0.5s  Figure 3.22. Persistence o f S T D in media containing low [Ca ] . Top traces: E P S C trains from a neuron during control, after a 1 m i n application o f 1.1 m M E D T A , after a 2 m i n wash i n 0.2 m M C a , and subsequent to a 1 m i n application o f 1% (vol/vol) D M S O . Lower traces, a x3 magnification o f the initial five E P S C s at the beginning o f the train are for E D T A and 0.2 m M C a . Bottom Left: E P S C amplitudes i n l o w C a media. Bottom middle: quantal size estimates. Bottom right: quantal content estimates. Data i n bottom plots are averages o f 5 neurons. Error bars i n 1 response indicate betweenneuron variations. e  +  2 +  2 +  st  Chapter 3. Results I Ran  - 110-  0.1 mM [Ca*] 2  0.5 nA 50 ms  3n  u 55  1 tog  O  a.  1.0H  CL UJ .  2  ***•#  0.5^  •  2  mM fCa*j  •  C.lmMjCa ] 1  •+*•*-§-•-«.-*-•  1  UJ  0.0  HiCa2+  Control  5  Lo Ca2+  10  20  15  stimulus #  th  Figure 3.23. [ C a ] modification o f S T D . Top: Traces showing rundown o f the 1 - 5 E P S C s i n 0.1 m M C a (light grey), 2 m M C a 2+ (dark grey), and 8 m M C a (black). Bottom Left: Scatter plot o f 1 E P S C amplitude in control (2 m M ) , high C a ( H i C a ; 8 2+ m M ) , and low C a (Lo C a ; 0.1 m M ) . Horizontal lines indicate mean. Bottom right: Normalized E P S C amplitudes in 0.1 m M C a (squares), 2 m M C a,2+ (circles), and 8 m M C a (triangles). Note increased amplitude after the intra-train gap i n the control and high [ C a ] and lack o f increase in amplitude in low C a . Data are from 10 neurons. Holdings potential was -80 m V . Values are expressed as Mean ± S E M . 2 +  st  e  2 +  z +  2 +  st  2 +  2 +  z +  2 +  z +  2 +  2+  2 +  2 +  - Ill -  Chapter 3. Results 1 Ran  Table 3.2.2A: Summary o f effects o f altered [Ca ] on parameters o f S T D e  Control (2 m M C a ) 2+  High C a (8mM)  2 +  Control Ca /EDTA ( 2 m M C a ) (0.1 m M C a ) 2 +  2 +  2+  Low C a (0.2mM)  2 +  Parameter Si(nA)  1.42 ± 0 . 4 1  2.53 ± 0 . 3 4  1.32 ± 0 . 5 3  0.47 ± 0.40  S (nA)  0.74 ± 0 . 1 9  1.42 ± 0 . 3 5  0.68 ± 0.27  0.34 ± 0 . 3 1  0.63 ± 0 . 1 9  S2/S1  0.52 ± 0 . 1 2  0.56 ± 0 . 1 3  0.51 ± 0 . 2 0  0.72 ± 0 . 1 6  0.68 ± 0 . 1 8  S12/S10  1.31 ± 0 . 0 8  1.56 ± 0 . 0 7 * 1.28 ± 0 . 1 1  1.03 ± 0 . 0 6  1.13 ± 0 . 0 5  Plateau  0.62 ± 0.09  0.91 ± 0 . 1 1 * 0.59 ± 0 . 1 3  0.13 ±0.10**  0.24 ± 0.08**  Plateau/Si  0.48 ± 0.05  0.34 ± 0.04* 0.44 ± 0.07  0.28 ± 0.04**  0.26 ± 0.06**  Apparent QN (nA)  3.93 ± 0 . 1 1  1.07 ± 0 . 2 1 * *  1.55 ±0.19**  2  0.92 ± 0.42  (Si 5-20) (nA)  7.2 ± 0.25  4.13 ± 0 . 1 7  Values are mean ± S E M between neurons; n = 5 i n high and low C a groups, * P < 0.05, Mest .** P< 0.05, A N O V A test. The controls were different neurons for high and low [ C a ] . 2+  e  Chapter 3. Results I Ran  112-  9+  Table 3.2.2B: Effects o f altered [Ca ] on derived parameters o f S T D e  Control (2mMCa ) 2 +  2+  High Ca (8 m M )  Control (2mM Ca ) 2+  Ca /EDTA (0.1mMCa ) 2 +  2 +  Low C a (0.2mM)  2 +  Parameter cov(S,,S ) 2  -0.018 ±0.010 -0.026 ±0.008 -0.021 ±0.013 -0.010 ±0.009 -0.015 ± 0 . 0 1 0  Q'(S0(pA)  33.4 ± 7 . 4  31.2 ± 8 . 6  35.4 ± 8 . 9  12. 6 ±8.6*  13.5 ±7.5*  Q'(S )(pA)  32.4 ± 6 . 5  29.5 ± 7.8  33.6 ± 7 . 5  11. 9 ±6.5*  13.1 ±4.2*  Q'(S,5-S ) (PA)  28.3 ± 6.9  26.9 ± 9 . 1  213 ±1.1  12.3 ± 7 . 0  14.2 ± 8 . 1  OYS^-S^ Q'(S,)  0.85 ± 0 . 1 7  0.87 ±0.11  0.78 ±0.21  0.97 ± 0 . 1 1  1.05 ± 0 . 1 8  Var/mean  29.4 ± 4.9  28.6 ± 6 . 6  27.9 ± 8 . 5  13.6 ± 9 . 2  14.6 ± 7 . 8  Evoked 30.8 ± 4.2 minEPSC size (pA)  32.7 ± 7 . 1  33.1 ± 5 . 7  14.5 ± 6 . 3  15.1 ± 6 . 0  13.2 ± 4 . 1  12.4 ± 3 . 7  13.9 ± 2 . 7  12. 7 ± 2 . 2  2  20  (S15-S20)  (PA)  Pre-stimulation minEPSC size 10.6 ± 2 . 9 (pA) mi  47 ± 9  78 ± 11*  37 ± 13  36 ± 19  68 ± 15  W15.20  26 ± 5  33 ± 8  19 ± 10  22 ± 7  17±6  m lm  1.21 ± 0 . 1 7  1.15 ± 0 . 1 1  1.02 ± 0 . 0 9  n  w  1.52 ± 0 . 0 9 *  1.13 ± 0 . 1 0  Values are mean ± S E M between neurons; n = 5 i n high and low [ C a ] groups; data from same neurons as Table 3.2.2A; * P < 0.05, Mest .** P< 0.05, A N O V A test. 2+  e  Chapter 3. Results I Ran  -113-  release, the changes i n S T D parameters occurred i n parallel to an increase i n quantal content at the beginning o f the train and around the intra-train gap (m^/mio) with no effect on quantal size (Table 3.2.2B). Hence, raising [ C a ] promoted S T D by increasing 2+  e  the quantal content, and increasing fractional release. The apparent increase i n Q N with 2*1"  high [ C a ^ e was also reported for neuromuscular junction (Elmqvist and Quastel, 1965a) and was consistent with a compound binomial model (Quastel, 1997) i n which fractional release varied between sites.  3.3.3. Receptor desensitization and saturation 3.3.3.1. Effects of blockade of receptor desensitization  The reduction i n quantal size observed during S T D (Figure 3.20B; Table 3.2.IB) suggested that a substantial component arises from receptor desensitization (Scheuss et al., 2002). Application o f a blocker o f A M P A receptor desensitization, cyclothiazide ( C T Z ; 50 u M ) had little effect on the development o f S T D (Figure 3.24; Table 3.2.3A), but estimates o f quantal size decreased less than i n controls reaching a higher plateau (Figure 3.24; Table 3.2.3B). These data demonstrated a moderate contribution o f receptor desensitization to the decrease in the apparent quantal size during S T D .  3.3.3.2. Combined blockade of receptor desensitization and saturation  To test whether receptor saturation, i n addition to desensitization, contributed to the early drop i n quantal size during S T D , C T Z (50 u M ) was co-applied with kynurenate ( K Y N ; 50 u M ) ; an antagonist that rapidly dissociates from glutamate receptors. The result was abolition o f the drop i n quantal size early i n the train (Figure 3.24C; Table 3.2.3B). A t the  -114-  Chapter 3. Results I Ran  A  Control |*|lll  CTZ  i  J/illii  'il'  !/ i/  A  0.5 ms  y /' '  0.5 nA  0.2 ms 0.5 nA  B  o  Control  •  Cyclothiazide(50 uM)  0 04 0.03  I cp. £• H  1 10  o  15  20  Control  10  15  20  25  0  10  15  20  Cyclothiazide (50 uM) * Kynurenate (50 uM)  1 VrTTTT!  Figure 3.24. Effects o f blockade o f receptor desensitization and saturation on S T D . A ) . Traces o f E P S C s (averages o f 5 repeats) before and after application o f 50 p M cyclothiazide ( C T Z ; 2 min). Traces on right show time expansions o f the l - 5 E P S C . B ) Left: Mean E P S C amplitudes (6 neurons) before and during application o f C T Z (50 p M ) . Note that the reduced depression resulted i n a higher plateau i n C T Z compared to control. Middle: quantal size estimates during C T Z application decreased to lesser extent compared to control reaching a higher plateau. Right: Quantal content estimates during C T Z application showed a decreased use-dependent reduction and a higher plateau. Note increase i n content subsequent to the intra-train gap. C ) Combined blockade o f receptor desensitization and saturation abolished use-dependent alterations i n quantal size but not S T D . Left: Mean E P S C amplitudes (6 neurons) before and during combined application o f C T Z (50 p M ) and K Y N (50 p M ) . Note the reduced amplitude o f the 1 E P S C which reached a plateau by the 5 response. Middle: quantal size estimates did not change during combined C T Z and K Y N application. Right: Quantal content estimates during combined C T Z and K Y N showed an increased use-dependent reduction and a lower plateau compared to control. Note increase i n content subsequent to the intra-train gap. Error bars i n 1 response show between neuron variations. (P < 0.001 i n amplitude data; P < 0.05 i n quantal size data; P < 0.01 i n quantal content data, Mests). s t  st  th  st  t h  Chapter 3. Results I Ran  - 115 -  Table 3.2.3A: Summary o f effects o f C T Z and K Y N on parameters o f S T D Control  CTZ (50 p M )  Control  CTZ + K Y N (50 p M ) (50 p M )  Parameter Si(nA)  1.29 ± 0 . 2 3  1.43 ± 0 . 2 6  1.21 ± 0 . 1 7  0.81 ± 0 . 1 3 *  S (nA)  1.04 ± 0 . 2 1  1.27 ± 0 . 2 2 0.97 ± 0 . 1 4  0.62 ± 0 . 1 4 *  s /s,  0.81 ± 0 . 1 3  0.73 ± 0 . 1 6 0.85 ± 0 . 1 0  0.88 ± 0 . 1 7  S12/S10  1.31 ± 0 . 1 5  1.23 ± 0 . 1 1  1.28 ± 0 . 1 9  1.21 ± 0 . 1 0  Plateau (Si 5-20) (nA)  0.59 ± 0 . 2 1  0.78 ± 0 . 1 9  0.55 ± 0 . 1 6  0.38 ± 0.24  Plateau/Si  0.45 ± 0 . 1 0 0.54 ± 0.09  0.45 ± 0 . 1 4  0.46 ± 0 . 1 7  Apparent Q N (nA)  3.93 ± 0.24  4.62 ± 0 . 3 1 * 3.99 ± 0 . 1 8  2.63 ± 0 . 4 1 *  2  2  Values are mean ± S E M between neurons; n = 6, * P < 0.05, Mest. Data are from 10 H z trains.  Chapter 3. Results I Ran  -116  Table 3.2.3B: Effects o f C T Z and K Y N on derived parameters o f S T D Control  CTZ  Control  (50 p M )  CTZ + K Y N (50 p M ) (50 p M )  Parameter cov(Si,S )  -0.027 ± 0 . 0 0 8  -0.022 ± 0 . 0 1 1  -0.024 ± 0 . 0 1 3  -0.011 ± 0 . 0 0 8  Q'(Si)(pA)  35.2 ± 2 . 5  38.3 ± 3.0  37.5 ± 5.7  25.3 ± 4.5*  Q'(S )(pA)  29.4 ± 6 . 4  37.2 ± 2.4  32.6 ± 3 . 9  26.1 ± 2 . 1 *  Q'(Si -S o) (pA)  26.1 ± 5 . 7  30.3 ± 3 . 1  27.3 ± 2.8  24.8 ± 3 . 1  OYSIS-STT,)  0.74 ± 0 . 1 5  0.79 ± 0 . 1 9  0.71 ± 0 . 1 3  0.98 ± 0 . 1 0 *  25.2 ± 5 . 1  2  2  5  2  Q'(Si) Var/mean (Sl5-S o) (PA)  27.2 ± 4 . 9  32.2 ± 4 . 1  29.4 ± 3 . 7  Evoked m i n E P S C size (pA)  32.0 ± 5 . 3  34.5 ± 3 . 9  31.0 ± 2 . 6  27.6 ± 4 . 6  Pre-stimulation m i n E P S C size (PA)  11.1 ± 3 . 4  12.3 ± 1 . 9  8.9 ± 2 . 4  2  m,  52 ± 8  OTI .  10.5 ± 2 . 2  53 ± 12  5 20  23 ± 5  35 ± 7  wi /mio  1.32 ± 0 . 2 1  1.16 ± 0 . 1 9  2  34 ± 14 24 ± 4 1.21 ± 0 . 1 8  37 ± 1 6 15 ± 5 1.28 ± 0 . 2 1  Values are mean ± S E M between neurons; n = 6 ; data from same neurons as Table 3.2.3A. * P < 0.05, Mest.  Chapter 3. Results 1 Ran  -117-  beginning o f the train, the quantal size decreased significantly compared to the control and remained unchanged throughout the train (Figure 3.24C; Table 3.2.3B). Despite a significant reduction i n the amplitudes o f the 1 and 2 st  n d  E P S C s , the characteristic E P S C  rundown during S T D (Plateau/Si) was unaffected by co-application o f C T Z and K Y N (Table 3.2.3 A ) . Given the lack o f effects on the quantal content, it was concluded that the effects o f co-applied K Y N and C T Z on S T D were predominantly postsynaptic.  The contribution o f receptor desensitization and saturation to S T D seems paradoxical since postsynaptic receptors cannot desensitize or saturate unless release has already occurred at a given site, whereas the negative covariances indicated that sites activated by the 2  n d  and 3  r d  stimuli are those that were not involved previously. This paradox could be  explained by an overflow o f transmitter from neighbouring release sites to receptor sites which were not previously activated (Telgkamp et al., 2004).  3.3.4. Effects of pentobarbital  on STD  3.3.4.1. EPSC behaviour in trains Pentobarbital enhanced S T D i n a dose-dependent manner i n the 2.5-20 H z stimulation range (Figure 3.25). The maximal enhancement o f S T D was at a concentration o f 200 pM which lowered the plateau producing a greater rundown o f E P S C s (Plateau/Si; Table 3.2.4A). A t this concentration, pentobarbital increased the ratio o f E P S C amplitudes around the intra-train gap (S12/S10; Table 3.2.4A). A parallel effect on the quantal content ratio around the intra-train gap (Table 3.2.4B), suggested an increase i n P . Pentobarbital 0  also produced an apparent reduction o f the product Q N (Table 3.2.4A). A correction o f  Chapter 3. Results I Ran  -118-  2.5 H z  10 H z  1.0O Control •  PB 50 uM •  PB 200 uM  o w  ft £  O Control •  PB 200 uM  PB 50 \iM  "8.3 N  0.5-  O. 0.5  to £ E «> 1 o z  0:0-  0.0 5  10  15  20  5  stimulus #  5 Hz PB 50 uM •  PB 200 uM  20  20 Hz  o w  1.0  N  PB 50 uM •  PB 200 uM  Q.  0.015  O Control •  ft-g IS E E «»•  0.5-  10  15  stimulus #  1.0O Control •  10  0.5  0.0  20  5  stimulus #  10  15  20  stimulus #  100!  on  10075-  to Ul  o.  50-  application time  10  time (min)  <D  o control  2 pM 20 pM * PB 50pM • PB 100 pM • PB200uM  50-  • PB  X5  PB 50 uM PB200 uM 15  A P B  25-  5  10  15  20  Stimulus frequency (Hz)  Figure 3.25. Dose-dependence o f pentobarbital enhancement o f S T D . A) Pentobarbital, applied at 50 and 200 u M , enhanced S T D producing a greater and faster rundown o f E P S C s with increasing stimulation frequencies. Pentobarbital did not abolish the post-gap increase i n the 12 E P S C amplitude. B) A decrease i n the amplitude o f the first E P S C amplitude was only observed after 4 m i n o f pentobarbital application. The data were obtained from samples within less than 4 m i n o f drug application. C) Dose dependence o f S T D at 2.5, 5, 10, and 20 H z . Pentobarbital, at 100-200 u M , enhanced S T D at 10 - 20 H z more than at lower stimulation frequencies (P < 0.05, A N O V A ) . Data represent averages obtained from 6 neurons. th  Chapter 3. Results I Ran  Table 3.2.4A: Summary o f pentobarbital effects on parameters o f S T D Pentobarbital concentrations (uM) Control  50  200  Si (nA)  1.21 ± 0 . 3 5  1.15 ± 0 . 2 9  1.11 ± 0 . 3 6  S (nA)  0.74 ± 0 . 1 9  0.66 ± 0 . 1 1  0.52 ± 0 . 1 3  S12/S10  1.20 ± 0 . 1 6  1.21 ± 0 . 2 1  1.67 ± 0 . 1 9 *  S2/S1  0.63 ± 0.07  0.58 ± 0.08  0.47 ± 0.05  Plateau  0.51 ± 0 . 1 0  0.34 ± 0.09  0.23 ± 0.07*  Plateau/Si  0.42 ± 0 . 1 2  0.30 ± 0.08  0.21 ± 0 . 0 7 *  Apparent Q N (nA)  4.31 ± 0 . 1 3  3.52 ± 0 . 2 1 *  2.70 ± 0 . 3 2 *  Parameter  2  (S15-S20) (nA)  Values are mean ± S E M between neurons; n = 6; * P < 0.05, A N O V A . Data are from 10 H z trains.  Chapter 3. Results I Ran  - 120-  •  Pentobarbital ( 5 0 | i M )  Pentobarbital ( 2 0 0 pM)  0.04 %  0.03  Ty-T-rf  B  (a) Before trains  Control  Pentobarbital (200 uM)  5 8 minis in 3 0 s •m- 1 0 . 1 ± 3 . 7 pA  1.0-  5 9 minis in 3 0 s 10.6 ± 3.2  ,0.8-  pA  j 0.6 iTo.4  foM  10  20 30 Size (pA)  40  10  5-  6 4 minis in 6 s  20 30 Size (pA)  48 minis in 6 s  _4  1  (b) After trains 0  10  31.8 ± 4.3  i  (b)-(a)  20 30 Size (pA)  pA  40  0  10  20 30 Size (pA)  40  ~, p-*i,  —  11.1 ± 3 . 6  pA  3-  'o Tib\~ 20 30 =  Size (pA)  0  10  20 30 Size (pA)  40  Figure 3.26. Quantal alterations mediated pentobarbital effects on S T D . A ) Left, apparent quantal sizes during corticothalamic S T D i n response to 10 H z stimulation. During application o f 200 u M pentobarbital, quantal sizes became significantly smaller starting at the 3 response until the end o f the train (P < 0.01, A N O V A test). Right: Effects o f pentobarbital on quantal contents during corticothalamic S T D . A low dose o f pentobarbital (50 (iM) decreased whereas a high dose increased the quantal content throughout the E P S C train. Note the significant increase i n quantal content after the omitted 1 1 stimulus. During application o f 50 or 200 uJVI pentobarbital, quantal content were significantly different than control starting at the 2 response until the end o f the train (P < 0.01, A N O V A test). Neurons held at -80 m V . Data obtained from 6 neurons. Error bars i n 1 response show between neuron variations. Jump after gap o f quantal content implied high P . B ) Pentobarbital reduced the amplitude o f evoked m i n E P S C s without affecting pre-stimulation m i n E P S C amplitude, (a) Histograms o f spontaneous m i n E P S C s obtained 5 s prior to the onset o f stimulation (6 repeats, 30 s i n total), (b) Histograms o f m i n E P S C s obtained 1 s after the end o f the stimulus train (6 repeats, 6 s i n total). Evoked m i n E P S C sizes were obtained after subtraction o f pre- from poststimulation evoked m i n E P S C s (b - a). Total m i n E P S C counts ('minis') are indicated above histograms. Values next to black arrows are mean size ± S E M . Data are from 1 neuron. r d  th  n d  st  0  Chapter 3. Results 1 Ran  121  Table 3.2.4B: Effect o f pentobarbital on derived parameters o f S T D Pentobarbital concentrations (pM) Control  50  200  0.025 ± 0 . 0 0 8  -0.031 ± 0 . 0 1 3  Parameter cov(S,,S ) 2  0.022 ± 0 . 0 1 1  Q'(Si)(pA)  35.2 ± 2 . 5  36.3 ± 3 . 0  29.6 ± 5 . 0  Q'(S ) (pA)  29.4 ± 6.4  31.3 ± 8 . 6  21.6 ± 7 . 4  Q'(Si5-s 2 o)  25.1 ± 5 . 7  19.8 ± 4 . 2  9.7 ± 5 . 1 *  0.71 ± 0 . 1 3  0.55 ± 0 . 1 6  0.37 ± 0 . 1 1  Var/mean (S15-S20) (pA)  27.2 ± 4.9  22.3 ± 4 . 2  13.5 ± 5 . 2  Evoked m i n E P S C size (PA)  32.0 ± 5 . 3  20.4 ± 8 . 1  11.3 ± 7.1  Pre-stimulation m i n E P S C size (pA)  11.1 ± 3 . 4  12.1 ± 3 . 1  10.8 ± 2 . 9  m\  34 ± 8  31 ± 5  41 ± 7  m 15-20  20 ± 7  17 ± 6  23 ± 7  2  (PA)  Q'(Si)  m /m\o u  1.18 ± 0 . 2 4  1.21 ± 0 . 1 9  1.85 ± 0 . 1 7  Values are mean ± S E M between neurons; n = 6 ; data from neurons o f Table 3.2.4 A ; * P < 0.05, A N O V A .  Chapter 3. Results I Ran  -122 -  Q N for a ((QN/l+a)) showed a similar effect o f pentobarbital ( a values were i n control 0.17 ± 0.03 and i n 200 p M pentobarbital 0.09 ± 0.02, P < 0.05, Mest; the corrected Q N values were 3.68 ± 0.17 n A i n control and 2.45 ± 0.26 n A i n 200 p M pentobarbital, P < 0.05, Mest). These effects were use-dependent and, hence, did not affect the 1 E P S C i n st  the train during application periods o f < 4 minutes (Figure 3.25 B ) .  W i t h pentobarbital, a major component o f S T D appeared to be a use-dependent reduction in quantal size (Figure 3.26; Table 3.2.4B). The ratio o f the plateau to the 1 apparent st  quantal size (Q'(S 15-20)/ Q'(Si)) decreased from 71 % in the control to 37% during pentobarbital application. The same effect was seen i n reduction i n the size o f evoked m i n E P S C without changes i n the spontaneous pre-stimulation m i n E P S C s (Figure 3.26; Table 3.2.4B). The modulation o f quantal parameters contingent on stimulation, and lack o f effects on pre-stimulation m i n E P S C s , suggested that pentobarbital produced smaller size quanta either by a presynaptic action o f selecting sites with small quanta, or a postsynaptic action confined to activated synaptic sites.  3.3.4.2. STD in raised Ca concentration 2+  The next set o f experiments examined i f pentobarbital effects on S T D could be modulated during conditions o f high release probability. A s previously, raising [ C a ] 2+  e  from 2 to 8 m M produced a greater rundown o f E P S C s . Under these conditions, pentobarbital (200 p M ) produced an even greater rundown, reducing the plateau to the 1  st  E P S C ratio (plateau/Si) to 0.19 ± 0.08 (P < 0.05, A N O V A ; Table 3.2.5A). A t this concentration o f pentobarbital, the amplitude ratio o f the E P S C s around the intra-train  Chapter 3. Results I Ran 94-  Table 3.2.5A: Pentobarbital effects on parameters o f S T D i n raised [Ca ] Control  High C a  (2mMCa ) 2 +  (8 m M )  Pentobarbital Pentobarbital 50 p M 200 p M (8 m M C a ) (8 m M C a ) 2+  2+  Parameter Si(nA)  1.11 ± 0 . 4 2  1.66 ± 0 . 3 5  1.59 ± 0 . 3 9  1.54 ± 0 . 4 1  S (nA)  0.69 ± 0 . 1 9  0.94 ± 0.23  0.71 ± 0 . 1 5  0.55 ± 0 . 2 1  S2/S1  0.63 ± 0 . 1 1  0.57 ± 0 . 1 4  0.45 ± 0 . 1 0  0.36 ± 0 . 1 6  S12/S10  1.20 ± 0 . 1 0  1.35 ± 0 . 0 8  1.38 ± 0 . 1 7  1.91 ±0.21***  Plateau  0.49 ± 0 . 1 0  0.58 ± 0 . 1 3  0.45 ± 0 . 1 5 0.27 ± 0.09**  2  (Si 5-20) (nA) Plateau/ S i  0.44 ± 0 . 1 0 0.36 ± 0 . 1 1  Apparent Q N (nA)  3.90 ± 0 . 1 3  0.28 ± 0.09  0.19 ± 0 . 0 8 *  5.35 ± 0 . 1 9 * 3.96 ± 0 . 2 1 * * 3.20 ± 0 . 1 7  ***  Values are mean ± S E M between neurons; n = 6; * (relative to control) ** (relative to high C a ) ***(relative to control and C a ) P < 0.05, A N O V A . Data are from 10 H z trains. 2+  2+  e  Chapter 3. Results I Ran  - 124 -  Table 3.2.5B: Pentobarbital effects on derived values o f S T D i n raised [Ca ] Control  H i g h Ca + 2  (2 m M C a ) 2+  cov(Si,S ) 2  (8 m M )  Pentobarbital 50 u M (8 m M C a ) 2+  0.017 ± 0 . 0 1 2 - 0.023 ± 0.009 -0.026 ± 0 . 0 1 3  e  Pentobarbital 200 u M (8 m M C a ) 2+  -0.031 ± 0 . 0 1 7  Q'(Si)(pA)  34.9 ± 8 . 1  35.4 ± 9 . 7  32.7 ± 8 . 1  30.5 ± 7.7  Q'(S ) (pA)  33.8 ± 9 . 2  32.6 ± 8.6  29.2 ± 7.3  24.7 ± 9 . 1  Q'(Si -S o) (pA)  29.4 ± 7.6  28.9 ± 6 . 9  25.6 ± 8 . 2  18.5 ± 10.3  OYS„-S,n) Q'(Si)  0.84 ± 0 . 1 4  0.81 ± 0 . 1 0  0.78 ± 0 . 1 2  0.60 ± 0.08*  Var/mean  30.8 ± 6 . 7  29.7 ± 8 . 5  27.6 ± 9 . 1  22.1 ± 7 . 9  33.1 ± 5 . 3  31.8 ± 9 . 4  28.3 ± 6.5  25.2 ± 8.4  Pre-stimulation 12.3 ± 2.9 m i n E P S C size (pA)  11.5 ± 4 . 3  10.6 ± 3 . 1  9.6 ± 3 . 7  2  5  2  (S15-S20)  (PA) Evoked m i n E P S C size (pA)  m\  31 ± 9  46 ± 11  48 ± 13  50 ± 15  m 15-20  16 ± 5  21 ± 8  17± 9  14 ± 13  mnlmxo  1.17 ± 0.13  1.36 ± 0 . 1 7  1.40 ± 0 . 1 2  1.95 ± 0 . 1 6 *  Values are mean ± S E M between neurons; n = 5 ; data from neurons o f Table 3.2.5A; * P < 0.05, A N O V A test.  Chapter 3. Results I Ran  - 125 2__ |  Table 3.2.6A: Pentobarbital effects on parameters o f S T D i n low [Ca ] Control  Low C a  (2 m M C a ) 2+  2 +  (0.1 m M )  Pentobarbital 50 p M (0.1 m M C a ) 2 +  e  Pentobarbital 200 p M (0.1 m M C a ) 2 +  Parameter Si(nA)  1.15 ± 0 . 4 2  0.41 ± 0.37  0.39 ± 0.22*  0.35 ± 0.27*  S (nA)  0.72 ± 0 . 3 1  0.29 ± 0.20  0.24 ± 0 . 1 9  0.19 ± 0 . 1 5 *  S2/S1  0.63 ± 0 . 1 4  0.73 ± 0 . 1 1  0.64 ± 0 . 1 2  0.50 ± 0 . 1 5  S12/S10  1.20 ± 0 . 1 1  1.01 ± 0 . 0 5  1.21 ± 0 . 0 8  1.67 ± 0 . 0 9 *  Plateau (Si 5-20) (nA)  0.50 ± 0 . 1 5  0.23 ± 0 . 1 1 *  0.19 ± 0 . 0 9 *  0.12 ± 0 . 1 0 *  Plateau/Si  0.43 ± 0.09  0.56 ± 0.06  0.48 ± 0 . 1 0  0.34 ± 0.08**  Apparent Q N (nA)  4.10±0.15  1.75 ± 0 . 2 4 *  1.43 ± 0 . 1 9 *  1.17 ± 0.15**  2  Values are mean ± S E M between neurons; n = 5, * (relative to control) ** (relative to low C a ) P < 0.05, A N O V A . Data are from 10 H z trains. 2+  Chapter 3. Results I Ran  -126 •  Table 3.2.6B: Pentobarbital effects on derived parameters o f S T D i n low [Ca Control  LowCa  2  e  Pentobarbital  Pentobarbital  50 u M (0.1mMCa )  200 u M (0.1mMCa )  (2mMCa )  (0.1 m M )  -0.023 ± 0 . 0 1 1  -0.014 ± 0 . 0 0 8 -0.017 ± 0 . 0 0 9 -0.021 ± 0 . 0 1 3  2 +  Parameter cov(S,,S )  2 +  ]  2 +  2 +  Q'(Si)(pA)  32.3 ± 9 . 1  14.2 ± 6 . 8 *  13.8 ± 8 . 8 *  12.7 ± 7.9*  Q'(S )(pA)  30.3 ± 8 . 1  13.7 ± 9 . 2 *  12.9 ± 7 . 5 *  12.1 ± 6 . 9 *  Q'(Si5-S o) (PA)  28.1 ± 7 . 7  14.4 ± 6 . 9  OYS^-STO)  0.87 ± 0 . 1 7  2  2  1.01  11.8 ± 8 . 3 *  9.4 ± 7.0*  0.74  ±0.11  **  ±0.13  0.85 ± 0 . 1 4  29.3 ± 9.0  14.5 ± 7 . 4  13.7 ± 8 . 2  11.5 ± 7 . 3 *  35.2 ± 6 . 1  15.1 ± 8 . 8  13.4 ± 5 . 1  10.8 ± 4 . 7 *  11.6 ± 4.1  Q'(Si) Var/mean (Si -S o) (PA) 5  2  Evoked minEPSC size (pA) Pre-stimulation m i n E P S C size (PA)  m  x  mi5- o 2  m lmxQ x2  11.7 ± 4.6  12.4±5.8  11.4 ± 3.7  35 ± 10  28 ± 12  29 ± 9  27 ± 13  16 ± 9  16 ± 7  12 ± 6  0.99 ± 0 . 1 2  1.21 ± 0 . 1 4  17 ± 8 1.19 ± 0 . 0 9  1.60 ± 0 . 1 1 * *  Values are mean ± S E M between neurons; n = 5 ; data from neurons o f table 3.2.6A; * (relative to control) ** (relative to low C a ) P < 0.05, A N O V A . 2+  Chapter 3. Results 1 Ran  -127 -  gap (S12/S10) nearly doubled. The reduction i n apparent Q N was similar to that observed 9+  in normal C a  (cf. Table 3.2.4A). Hence, pentobarbital effects on S T D were additive to  the increased E P S C rundown caused by raised C a . 2 +  Raising [Ca ] did not greatly alter pentobarbital effects on quantal size and content e  (Table 3.2.5B). In raised [ C a ] , pentobarbital decreased the ratio o f the plateau to the 1 2+  st  e  apparent quantal size and increased the ratio o f the quantal contents around the intra-train gap, similar to the effects observed i n 2 m M [ C a ] (Table 3.2.5B, cf. Table 3.2.4B). 2+  e  However, reduction i n Q' was much less than in 2 m M [ C a ] . 2+  e  3.3.4.3. STD in reduced Ca concentration 9+  Reducing [Ca ] from 2 to 0.1 m M did not affect the pentobarbital enhancement o f S T D e  (Table 3.2.6A, B ) . In low [ C a ] , pentobarbital still produced a greater rundown then it 2+  e  did i n normal [ C a ] media. The effects o f low [ C a ] to reduce the apparent quantal 2+  2+  e  size, variance-mean ratio, and evoked m i n E P S C  e  size were  farther  modified  by  pentobarbital, which caused further rundown i n Q', and an increased ratio o f the quantal contents around the intra-train gap. This unexpected latter effect implicates a presynaptic action on the high release pool o f quantal packets which mediate S T D i n l o w [ C a ] 2+  e  media. In summary, pentobarbital enhancement o f S T D was resistant to reductions i n [Ca ] . 2+  e  Chapter 3. Results I Ran  - 128 -  3.3.4.4. Combined cyclothiazide and kynurenate blockade In order to unmask the presynaptic actions o f pentobarbital, it was necessary to reduce the postsynaptic contributions to S T D . In other neurons, pentobarbital has been reported to have postsynaptic actions o f promoting A M P A receptor desensitization (Jackson et al., 2003), which would contribute to S T D . Receptor saturation is also a possible contributor to S T D (Chen et al., 2002). For these reasons, pentobarbital actions on S T D were reexamined during pharmacological blockade o f receptor desensitization and saturation.  Pentobarbital enhancement o f S T D was unaffected by a combined blockade o f receptor desensitization and saturation. During co-application o f C T Z (50 p M ) with K Y N (50 p M ) , pentobarbital (200 p M ) still increased the rundown o f E P S C s i n a use-dependent manner (Figure 3.27; Table 3.2.7A). The apparent quantal size plateau (Q'(S 15-20)), ratio o f Q'(Si5-S2o)/ Q ' S i , variance-mean ratio, and evoked m i n E P S C size all decreased i n response to pentobarbital (Table 3.2.7B). In addition, the quantal content plateau (m\s-2o) also increased significantly relative to control or co-applied C T Z + K Y N (Table 3.2.7B). The above data implicated an enhancement o f S T D by a presynaptic action that reduced the quantal size.  3.3.5. Effects of altered extracellular concentration ([Jt] ) on STD e  Transmitter release is sensitive to [ K ] alterations, which modify the membrane potential +  e  in the nerve terminal (Hatt and Smith 1976; Saint et al. 1987). This provided a rationale to examine the effects o f altering [ K ] on S T D and their modulation by pentobarbital. +  e  Chapter 3. Results I Ran  - 129-  O Control •  o4  0  ,  5  1  •  Cyclothiazide (50 pM) + Kynurenate (50 p.M)  Cyclothiazide (50 pM) + Kynurenate (50 pM) +Pentobarbital (200 pM)  ,  10 15 stimulus #  ,  20  o.oo-l  0  ,  5  ,  ,  10 15 stimulus #  ,  20  o-l  1  0  5  ,  1  10 15 stimulus #  ,  20  Figure 3.27. Pentobarbital enhancement o f S T D during combined blockade o f receptor desensitization and saturation. Co-application o f C T Z (50 uJVI) with K Y N (50 u M ) reduced the amplitude o f E P S C s (left) and abolished the decrease i n quantal size throughout the train (middle). The quantal content (right) had a time course similar to that o f E P S C amplitude (left) i n the presence o f co-applied C T Z and K Y N . Under these conditions, pentobarbital application enhanced E P S C depression (left) and reduced the quantal size (middle) throughout the duration o f E P S C train. In parallel, pentobarbital increased the quantal content (right) during the train at a rate similar, and possibly due to the decrease i n quantal size. Data are from 5 neurons. Holding potential was -80 m V . Error bars o f 1 response show between neuron variations. st  Chapter 3. Results I Ran  -130 -  Table 3.2.7A: Pentobarbital effects on parameters o f S T D during co-applied C T Z and KYN Control  CTZ + K Y N (50 p M ) (50 p M )  Pentobarbital + C T Z + K Y N (200 p M ) (50 p M ) (50 p M )  Parameter S,(nA)  1.29 ± 0 . 1 7  0.89 ± 0 . 1 3 *  0.85 ± 0 . 1 4 *  S (nA)  1.04 ± 0 . 1 3  0.62 ± 0 . 1 4 *  0.60 ± 0 . 1 8 *  S2/S1  0.85 ± 0 . 1 0  0.88 ± 0 . 1 7  0.70 ± 0 . 1 1  S12/S10  1.28 ± 0 . 1 9  1.21 ± 0 . 1 0  1.15 ± 0.12  Plateau  0.61 ± 0 . 1 6  0.49 ± 0 . 1 4  0.34 ± 0 . 1 1  Plateau/Si  0.47 ± 0.08  0.55 ± 0.05  0.40 ± 0.06**  Apparent Q N (nA)  3.99 ± 0 . 1 8  2.63 ± 0 . 4 1  3.42 ± 0.27  2  (S15-20) (nA)  Values are mean ± S E M between neurons; n = 6;* (relative to control) ** (relative to C T Z + K Y N ) P < 0.05 A N O V A test. Data are from 10 H z trains.  Chapter 3. Results I Ran  -131 -  Table 3.2.7B: Pentobarbital effects on derived parameters o f S T D during co-applied C T Z and K Y N Control  CTZ + K Y N (50 u M ) (50 u M )  Pentobarbital + C T Z + K Y N (200 u M ) (50 u M ) (50 u M )  Parameter cov(Si,S ) 2  -0.024 ± 0 . 0 1 3 -0.011 ± 0 . 0 0 8  -0.019 ± 0 . 0 1 1  Q'(Si)(pA)  37.5 ± 5 . 7  25.3 ± 4.5  Q'(S )(pA)  32.6 ± 3 . 9  26.1 ± 2 . 1 *  17.6 ± 1 . 9 *  Q'(Si5-S ) (PA)  27.3 ± 2 . 8  24.8 ± 3 . 1  4.1 ± 3 . 8  O'fSiyS™) Q'(Si)  0.71 ± 0 . 1 3  0.98 ± 0 . 1 0  0.17 ± 0 . 1 1 *  Var/mean (Sl5-S o) (PA)  29.4 ± 3 . 7  Evoked m i n E P S C size (pA)  31.0 ± 2 . 6  27.6 ± 4 . 6  10.3 ± 3 . 8 *  Pre-stimulation m i n E P S C size (PA)  12.3 ± 1 . 9  8.9 ± 2.4  9.8 ± 2 . 1  mi  34 ± 14  37 ± 16  36 ± 1 0  /ni _2o  24 ± 4  15 ± 5  78 ± 9 *  2  20  25.2 ± 5 . 1  24.5 ± 5.3  6.9 ± 4 . 4 *  2  5  mn/mio  1.21 ± 0 . 1 8  1.28 ± 0 . 2 1  1.09 ± 0 . 1 7  Values are mean ± S E M between neurons; n = 6 ; data from same neurons as Table 3.2.7A; * (relative to control) ** (relative to C T Z + K Y N ) P < 0.05, A N O V A .  Chapter 3. Results I Ran  - 132 -  3.3.5.1. High [K^Je perfusion Raising [ K ] from 2.5 to 10 m M caused significant changes i n the shape o f S T D (Figure +  e  3.28A; Table 3.2.8A). The amplitude o f the 2  n d  E P S C facilitated relative to the 1  st  EPSC  (Figure 3.28A). The E P S C plateau increased whereas the rundown (Plateau/Si ratio) was raised compared to control [ K ] conditions (Table 3.2.8A). The apparent Q N nearly +  e  doubled i n response to high [ K ] application (Table 3.2.8A), indicating an enhancement +  e  o f refill or recruitment o f sites previously with low fractional release, now to be included i n the releasable pool (Quastel, 1997).  Unexpectedly, high [ K ] application abolished the covariance between the 1 +  st  e  and 2  n d  E P S C , yielding a small positive value (Table 3.2.8B). This might or might not be a statistical aberration. The apparent quantal  size, variance-mean  ratio and evoked  m i n E P S C size were unaffected by high [ K ] (Table 3.2.8B). These data implicated a +  e  presynaptic action that increased the fractional release and pulse to pulse facilitation.  In 10 m M [ K ] , pentobarbital had a marked effect on the shape o f E P S C train. +  e  Pentobarbital effects included: 1) transforming the facilitation between the 1  st  and 2  n d  E P S C into depression; 2) decreasing the plateau; and, 3) increasing the rundown o f E P S C s , (Figure 3.28 A ; Table 3.2.8A). The apparent Q N also decreased, similar to the effects i n normal [ K ] media (cf. Table 3.2.4A). A similar effect o f pentobarbital was +  e  observed, after correcting Q N for a values (QN/(l+a); a values were 0.15 ± 0.04 i n control, 0.16 ± 0.02 i n high [ K ] , and 0.12 ± 0.03 i n pentobarbital/high [ K ] (P > 0.05, t+  +  test). The corrected Q N values were (in n A ) 5.11 ± 0.22 i n control, 9.38 ± 0.41 i n high  Chapter 3. Results I Ran  - 133 -  [ K ] , and 5.04 ± 0.37 i n pentobarbital and high [ K ] (P < 0.05, Mest). Pentobarbital also +  +  e  e  restored the negative covariance at the beginning o f the train. The use-dependent effects o f pentobarbital on the decline o f the apparent quantal size early i n the train (QYJS15S2o)/Q'(Si) ratio) were similar to the effects in normal [ K ] (Table 3.2.8B). The total +  e  pentobarbital suppression o f the increase in S2/S1 produced by 10 m M [ K ] indicated a +  e  presynaptic effect o f pentobarbital distinct from what was seen i n other experiments.  The apparent increase i n Q N by 10 m M K reflects the raised S2, S3, S4 +  presumably  reflecting a combination o f high a and rising P . The unchanged S i i n high [ K ] +  0  e  indicated that Q N (~6 n A ) did not change at the beginning o f the train. The data indicate fractional release o f about 0.3 for S i and S2 under control conditions, with 10 m M [ K ] +  e  raising fractional release to about 0.9 or more at S2. Hence, hypothetically, fractional release could have increased greatly i f a after the first pulse remained low, or could have remained nearly unchanged, i f a were to become near 1, as suggested by the absence o f negative covariance, or anywhere in between, depending upon how a is postulated to have changed.  3.3.5.2. Low [Kf]  e  perfusion  Reducing [ K ] concentration did not affect S T D (Table 3.2.9 A , B ) . However, the mean +  e  ratio o f E P S C amplitudes and quantal contents around the intra-train gap decreased compared to normal [ K ] (Table 3.2.9A, B ) . This observation suggested that the post-gap +  e  increases i n E P S C size and quantal content were attenuated as a result o f the low [ K ] +  e  -134-  Chapter 3. Results I Ran  B  PB 200 uM (0.1 mM K)  Control  1  0.1 mM K  (2.5 mM K*)  +  pWtt 1/nA 0.5s  ©Control  ;._,_:.:.:::_^ 0  5  10  • L o w K'  Q.OO I  15 .  20  o  stimulus #  t  5  ...  • PB (Low K ) +  . .!  10:  :  15  0i  ,• 20  •  -  0  • 5  stimulus*  T  T  "  10  "  t 15  1  •' 20  .stimulus.*!  Figure 3.28. Effects o f altered [ K ] on pentobarbital enhancement o f S T D . A) Effects o f raised [ K ] . Top: E P S C traces during application o f 2.5 m M [ K ] (left), 10 m M [ K ] (middle), and 200 u M pentobarbital i n 10 m M [ K ] (right). Bottom: Mean E P S C amplitudes (left), quantal size estimates (middle), and quantal contents (right) from control, raised [ K ] , and pentobarbital i n raised [ K ] . Data are from 5 neurons. B) Effects o f reduced [ K ] . Top: E P S C traces during application o f 2.5 m M [ K ] (left), 0.1 m M [ K ] (middle), and 200 u M pentobarbital i n 0.1 m M [ K ] (right). Bottom: Mean E P S C amplitudes (left), quantal size estimates (middle), and quantal contents (right) from control, reduced [ K ] , and pentobarbital i n reduced [ K ] . Traces i n A and B are responses to single trains from one neuron each. Other data are averages from 5 neurons. Error bars i n 1 response show between-neuron variations. +  e  +  +  +  e  e  e  +  e  +  +  e  +  +  e  e  +  +  e  +  +  e  st  e  - 135-  Chapter 3. Results I Ran  Table 3.2.8A: Summary o f effects o f high [ K ] , pentobarbital on parameters o f S T D +  e  Control (2.5 m M K ) +  High K (10 m M ) +  Pentobarbital 200 p M (in high K ) +  Parameter Si(nA)  1.78 ± 0 . 2 5  1.75 ± 0 . 1 2  1.73 ± 0 . 1 1  S (nA)  1.08 ± 0 . 1 5  1.92 ± 0 . 1 9 *  1.24 ±0.14**  S2/S1  0.60 ± 0 . 1 1  1.08 ± 0 . 1 7 *  0.71 ±0.15**  S12/S10  1.31 ± 0 . 1 0  1.05 ± 0 . 1 4 *  1.45 ±0.17**  Plateau (Si 5-20) (nA)  0.67 ± 0 . 1 5  0.93 ± 0 . 1 9 *  0.52 ±0.16**  Plateau/Si  0.38 ± 0 . 1 0  0.53 ± 0 . 1 9 *  0.31 ± 0 . 2 1 * *  Apparent Q N (nA)  5.91 ± 0 . 2 5  10.83 ± 0 . 3 3 *  5.65 ± 0.47**  2  Values are mean ± S E M between neurons; n = 5; * (relative to control) ** (relative to high K ) P < 0.05 A N O V A test. Data are from 10 H z trains. +  Chapter 3. Results I Ran  - 136 -  Table 3.2.8B: Derived parameters o f S T D in high [ K ] , pentobarbital +  e  Control (2.5 m M K ) +  High K (10 m M )  +  Pentobarbital 200 p M (in 10 m M K ) +  Parameter cov(Si,S )  -0.017 ± 0 . 0 0 7  0.003 ± 0.005*  Q'(Si)(pA)  34.1 ± 7 . 4  33.8 ± 3 . 0  Q*(S )(pA)  29.0 ± 6 . 9  28.7 ± 2 . 9  28.3 ± 2.6  Q'(Si5-S ) (pA)  28.3 ± 6 . 1  29.9 ± 1 0 . 8  18.0 ± 7 . 1  OYSis-SW) Q'(Si)  0.83 ± 0 . 1 0  0.88 ± 0 . 1 7  0.53 ± 0.09*  Var/Mean (Si5-S rj) (PA)  30.1 ± 4.4  32.3 ± 5.1  Evoked m i n E P S C size (pA)  32.0 ± 5 . 3  31.8 ± 6 . 6  Pre-stimulation m i n E P S C size (PA) m,  11.1 ± 3 . 4  12.3 ± 4.1  2  2  20  -0.011 ± 0 . 0 0 9 33.6 ± 2 . 5  20.3 ± 6.2  2  24.3 ± 4 . 2  10.9±3.7  54 ± 8  52 ± 6  79 ± 1 0  m .2o  23 ± 3  32 ± 4  44 ± 6  m /m  1.32 ± 0 . 0 7  1.09 ± 0 . 0 9  1.15 ± 0 . 1 1  l5  n  l0  Values are mean ± S E M between neurons; n = 5 ; data from same neurons as table 3.2.8A; * (relative to control) ** (relative to high K ) P < 0.05 A N O V A test. +  Chapter 3. Results I Ran  Table 3.2.9A: Summary o f effects o f low [ K ] , pentobarbital on parameters o f S T D +  e  Control (2.5 m M K ) +  Low K (0.1 m M ) +  Pentobarbital 200 p M (in 0.1 m M K ) +  Parameter Si(nA)  1.65 ± 0 . 2 0  1.60 ± 0 . 1 7  1.55 ± 0 . 2 5  S (nA)  1.10 ± 0.17  0.99 ± 0 . 1 0  1.42 ± 0 . 1 5  s /s,  0.66 ± 0 . 1 3  0.40 ± 0 . 1 8  0.91 ± 0 . 2 5  Si /Sio  1.21 ± 0 . 0 7  1.01 ± 0 . 0 9  1.40 ± 0.11*  Plateau (Sl5- fj) (nA)  0.67 ± 0 . 1 8  0.60 ± 0 . 1 1  0.39 ± 0 . 1 7  Apparent Q N (nA)  5.90 ± 0 . 3 0  2  2  2  2  5.25 ± 0.23  6.25 ± 0.43*  Values are mean ± S E M between neurons; n = 5; * (relative to low K ) P < 0.05, A N O V A test. Data are from 10 H z trains. +  Chapter 3. Results I Ran  - 138  Table 3.2.9B: Derived parameters o f S T D in low [ K ] , pentobarbital +  e  Control (2.5 m M K ) +  Low K (0.1 m M ) +  Pentobarbital 200 p M (in 0.1 m M K ) +  Parameter cov(Si,S )  -0.019 ± 0 . 0 0 9  Q'(Si)(pA)  33.4 ± 2 . 5  33.0 ± 11.3  32.3 ±  Q'(S )(pA)  29.5 ± 2 . 0  28.7 ± 9 . 5  27.8 ± 1 2 . 1  Q'(Si5-S ) (pA)  28.5 ± 5 . 7  28.0 ± 8 . 1  21.8 ± 6 . 9  OYSis-S?^ Q'(S.)  0.85 ± 0 . 1 2  0.84 ± 0 . 1 9  0.67 ± 0 . 1 1  Var/Mean (Si5-S o) (pA)  31.4 ± 6 . 1  30.5 ± 7.0  23.4 ± 8 . 2  Evoked m i n E P S C size (PA)  30.1 ± 4.1  29.7 ± 8.0  26.1 ± 5.1  9.7 ± 2 . 4  10.1 ± 3 . 2  9.9 ± 3 . 9  54 ± 9  50 ± 5  51 ± 8  23  21  17  2  2  20  -0.015 ± 0 . 0 0 7  -0.009 ± 0 . 0 0 6 8.9  2  Pre-stimulation m i n E P S C size (pA) wi W15-20  m /m l2  l0  ±9  1.33 ± 0 . 2 3  ±6  1.04 ± 0 . 1 6  ±6  1.43 ± 0 . 1 4 *  Values are mean ± S E M between neurons; data from same neurons as table 3.2.9A; n = 5, * (relative to low K ) P < 0.05 A N O V A test. +  Chapter 3. Results I Ran  - 139 -  contrary to effect o f nerve terminal hyperpolarization at the neuromuscular junction which would theoretically increase release (Hubbard et al., 1967).  In low [ K ] , pentobarbital had few effects on S T D (Table 3.2.9A, B ) . The sole +  e  significant effect was to increase the post-gap jump i n E P S C amplitude (Table 3.2.9A). This effect is compatible with a raised fractional release (P ), raised P -(l-oc), and/or 0  0  lowered a, and occurred i n conjunction with increases i n the quantal content (Figure 2 8 B , Table 3.2.9B). Hence, reducing [ K ] revealed release-promoting actions o f pentobarbital, +  e  distinct from those observed i n normal or raised [ K ] . +  e  3.3.6. Effects of tetrodotoxin (TTX) The following investigations examined the effects o f partial blockade o f voltage-gated N a channels on S T D . Application o f 8 to 64 n M T T X caused significant changes i n the +  configuration o f S T D (Figure 3.29). A t 32 n M , T T X reduced the 1  st  and 2  n d  EPSC  amplitudes and increased the rundown o f the E P S C train (Figure 3.29; Table 3.2.1 OA). A t this concentration, T T X also significantly reduced the apparent Q N . Unexpectedly, the ratio o f E P S C s around the intra-train gap increased to a value > 2. This observation was in contrast to the prediction o f the binomial model that the amplitude o f the E P S C following the intra-train gap could increase only to twice as much as the previous E P S C amplitude. This result suggested that some blockade o f presynaptic action potentials reversed the increased gap between stimuli. A t 64 n M , T T X irreversibly abolished E P S C s (Figure 3.29), as expected i f presynaptic action potentials were completely blocked.  Chapter 3. Results IRan  -140-  T T X effects on S T D also involved a use-independent reduction i n quantal size i n the entire E P S C train (Figure 3.29; Table 3.2.1 OB). The decrease i n the apparent quantal size was significant already at the 1 and 2 st  n d  responses (Q'(Si), Q'(S2))- The plateau o f the  apparent quantal size (Q'(Si5-2o)) and its ratio to the 1 quantal size (Q'(Si5-S o)/ Q'(Si)) st  2  also decreased significantly during T T X (Table 3.2.1 OB). These effects o f T T X coincided with reductions i n the variance-mean ratio and the evoked m i n E P S C size (Figure 3.30;  Table 3.2.1 OB). The amplitude o f pre-stimulation m i n E P S C s was unaffected by T T X application, arguing against a postsynaptic action. Hence, T T X effects on S T D likely involved a presynaptic use-dependent action that produced smaller quanta, plus an effect to reduce quantal size that did not reverse i n the 20 s between-train period.  In order to unmask the net presynaptic actions o f T T X , it was necessary to re-examine its effects i n conditions that reduce postsynaptic contributions to S T D . For this reason, the experiments were repeated with co-applied C T Z and K Y N . A s previously observed, coapplication o f C T Z (50 p M ) with K Y N (50 p M ) decreased the 2  n d  and plateau E P S C  amplitudes as well as the apparent Q N (cf. Table 3.2.3A). A subsequent application o f T T X (32 n M ) produced a lower E P S C plateau and apparent Q N values and a faster E P S C rundown, similar to the effects without co-applied C T Z + K Y N (cf. Table 3.2.10A).  In these experiments, T T X affected quantal parameters nearly the same way as without co-applied C T Z and K Y N (cf. Table 3.2.10B). The apparent quantal size decreased throughout the train (Table 3.2.1 IB). The plateau o f apparent quantal size, variance-mean  Chapter 3. Results 1 Ran  Control Mtyfmrr'yrcrmi  -141 -  TTX 8 nM  TTX 32 nM  TTX 64 nM  0.5 s 0.5 nA  B  o control • TTX 8nM • TTX 32 nM  "g  1.0  Q.  E to  a  0.5  i+iHi Wr%i  *4  ^  stimulus #  Figure 3.29. Tetrodotoxin enhanced S T D by reducing quantal size. A ) Traces o f E P S C trains during application o f T T X at 8, 32, and 64 n M . A t 8 - 32 n M T T X application enhanced S T D producing a greater and faster rundown o f E P S C s . A t 32 n M , T T X decreased the amplitude o f the E P S C s early i n the train. Application o f T T X at 64 n M resulted i n irreversible loss o f E P S C s . B ) Left: Mean E P S C amplitude from 6 neurons before and during application o f 8 and 32 n M T T X . Middle: Apparent quantal size estimates during S T D . A t 32 n M , T T X reduced the quantal size significantly (P < 0.01, ttest). The quantal size decreased and reached a plateau at response 4. Right: Quantal content during T T X enhancement o f S T D . Note the increase i n quantal content following the intra-train gap. Data are from 5 neurons. Holding potential was - 80 m V . Error bars o f first response show between-neuron variations.  Chapter 3. Results I Ran  -142-  TTX 32 nM  Control Before trains  59 minis in,3Q s 11.2 ± 2.3 pA  66 minis In 30 «'  ~f\\  10.9 • 2.7 pA  Mr f o .  0  10  20 30 Size (pA)  40  ;0  10  B  20 30 Size (pA)  40  After trains 69 minis in 6 s  38 minis in 6 s  -*4  I' | &i 3  2  0  10  20 30 Size (pA)  40  0  10  20 30 Size (pA)  40  B -A 32:9 ± 2 . 2 pA  i. 0'  1.10,  20 30 Size(pA)  40  4 3  12.4 ± 3 . 4 pA  -V 0  10  20 30 Size(pA)  40  Figure 3.30. T T X decreased the size o f evoked miniature E P S C s without affecting spontaneous miniature E P S C size. A ) Amplitude histograms o f spontaneous m i n E P S C s 5 s before the stimulus train (6 repeats, 30 s i n total). B ) Amplitude histograms o f evoked m i n E P S C s counted 1 s after the stimulus train (6 repeats, 6 s total). Total mini count is indicated above histograms. Evoked m i n E P S C sizes were obtained after subtraction o f spontaneous from evoked m i n E P S C histogram (B - A ) . Values next to black arrows pointing at peaks are mean size ± S E M . Data are from 1 neuron.  Chapter 3. Results I Ran  Table 3.2.10A: Summary o f T T X effects on parameters o f S T D T T X concentrations (nM) Control  8  32  Parameter Si (nA)  1.19 ± 0 . 3 3  0.95 ± 0.36  0.49 ± 0.28*  S (nA)  0.87 ± 0 . 1 4  0.80 ± 0 . 3 4  0.40 ± 0 . 1 6 *  S2/S1  0.73 ± 0.07  0.84 ± 0.09  0.81 ± 0 . 1 1  S12/S10  1.51 ± 0 . 1 1  1.40 ± 0 . 1 9  2.52 ± 0 . 4 1 *  Plateau (Si 5-20) (nA)  0.39 ± 0.08  0.20 ± 0 . 1 2  0.12 ± 0 . 0 9 *  Plateau/ S i  0.32 ± 0 . 1 1  0.21 ± 0.07  0.24 ± 0.08  Apparent Q N (nA)  4.40 ± 0 . 1 7  4.59 ± 0 . 3 1  1.82 ± 0 . 3 5 *  2  Values are mean ± S E M between neurons; n = 5, * P < 0.05, A N O V A . Data are from 10 H z trains.  - 144-  Chapter 3. Results I Ran  Table 3.2.10B: T T X effects on derived parameters o f S T D T T X concentrations (nM) Control  8  32  Parameter 0.026 ± 0 . 0 1 2  -0.019 ± 0 . 0 1 3  0.014 ± 0 . 0 0 9  Q'(Si) (pA)  33.5 ± 2 . 5  34.6 ± 6.3  17.8 ± 5 . 1 *  Q'(S )(pA)  27.8 ± 2.0  30.0 ± 5 . 3  14.2 ± 3 . 6 *  Q'(Si5-S ) (PA)  24.5 ± 3.6  19.8 ± 4 . 3  7.9 ± 3 . 8 *  QXSJS^Q)  0.73 ± 0 . 1 1  0.57 ± 0.09  0.44 ± 0.07*  27.8 ± 4.7  22.9 ± 5 . 1  cov(S,,S ) 2  2  20  Q'(Si) Var/Mean (Si5-S o) (PA)  9.6 ± 2 . 8 *  2  Evoked m i n E P S C size (pA)  32.1 ± 4 . 1  Pre-stimulation minEPSCsize (PA)  10.1 ± 2 . 0  22.3 ± 3.0  10.7 ± 1.6  12.9 ± 3 . 3 *  9.8 ± 1.9  m\  35 ± 6  27 ± 10  28 ± 9  m 15-20  18 ± 3  10±7  16±4  1.52 ± 0 . 2 5  1.42 ± 0 . 3 1  mn/mio  2.52 ± 0.27*  Values are mean ± S E M between neurons; data from same neurons as Table 3.2.1 OA; n = 5, * P < 0.05, A N O V A .  Chapter 3. Results I Ran  -145-  O control  • CTZ+KYN  • CTZ+KYN+TTX  "?7"T r r r r r r r r T  N-T f  10 stimulus #  15  8 75 40-  fTT*T 20  5  10 stimulus #  15  20  Figure 3.31. T T X effects on S T D during blockade o f receptor desensitization and saturation. Co-application o f C T Z (50 m M ) with K Y N reduced the amplitude o f E P S C s without affecting the rundown (left). Under these conditions, the quantal size (middle) did not change throughout the train whereas the quantal content (right) followed the time course o f the E P S C train. A subsequent application o f T T X (32 n M ) enhanced S T D reducing the plateau and increasing the rundown. T T X decreased the quantal size, already at the 2 response and increased the quantal content, from the 8 response until the end o f the train (right). Data are from 5 neurons. Holding potential was -80 m V . Error bars o f 1 response show between-neuron variations. n d  st  th  Chapter 3. Results I Ran  -146-  Table 3.2.11 A : T T X effects on parameters o f S T D during co-applied C T Z + K Y N Control  CTZ + K Y N (50 p M ) (50 p M )  TTX + CTZ + K Y N (32 n M ) (50 p M ) (50 p M )  Parameter Si(nA)  1.29 ± 0 . 2 4  0.85 ± 0 . 1 8  0.83 ± 0 . 1 6 *  S (nA)  1.04 ± 0 . 1 3  0.62 ± 0 . 1 0 *  0.51 ± 0 . 1 1 *  S2/S1  0.80 ± 0 . 1 0  0.73 ± 0.07  0.61 ± 0;09  S12/S10  1.21 ± 0 . 1 3  1.15 ± 0 . 0 9  1.22 ± 0 . 0 8  Plateau  0.62 ± 0 . 1 0  0.38 ± 0 . 1 1 *  0.22 ± 0 . 1 2 *  Plateau/Si  0.48 ± 0.09  0.44 ± 0.06  0.26 ± 0.05**  Apparent Q N (nA)  5.04 ± 0.35  3.04 ± 0.25*  2.5 ± 0 . 3 1 * *  2  (Si  5-20)  (nA)  Values are mean ± S E M between neurons; n = 5, * (relative to control) ** (relative to control and C T Z + K Y N ) P < 0.05, A N O V A . Data are from 10 H z trains.  Chapter 3. Results 1 Ran  - 147-  Table 3.2.1 I B : T T X effects on derived parameters o f S T D during co-applied C T Z and KYN Control CTZ + K Y N TTX + CTZ + K Y N (50 p M ) (50 p M )  (32 n M ) (50 p M ) (50 p M  Parameter cov(Si,S ) 2  -0.026 ± 0 . 0 1 0 -0.013 ± 0 . 0 0 7  -0.018 ± 0 . 0 1 3  Q'(S0(pA)  37.0 ± 6 . 1  24.7 ± 5 . 1 *  25.6 ± 4.3*  Q'(S )(pA)  32.6 ± 5 . 0  25.6 ± 3 . 7  19.1 ± 4 . 3 *  Q'(Si5-S ) (PA)  27.1 ± 3 . 7  24.8 ± 5 . 1  2  20  OYSis-S^ Q'(S.)  0.73 ± 0 . 1 9  Var/mean (Sl5-S n) (PA)  5.1 ±2.9**  1.03 ± 0 . 1 1 *  0.20 ± 0 . 1 3 * *  29.4 ± 3 . 3  26.1 ± 2 . 8  7.2 ± 2.5  Evoked m i n E P S C size (pA)  32.0 ± 5 . 3  27.6 ± 4 . 2  9.4 ± 3 . 7 **  Pre-stimulation m i n E P S C size (PA)  11.1 ± 3 . 4  10.9 ± 2 . 4  2  mi  34 ± 1 1  m.  l5 20  wi /wio 2  23 ± 9 1.23 ± 0 . 1 2  33 ± 14  15 ± 8 1.27 ± 0 . 1 4  10.4 ± 3 . 1  30 ± 9  50 ± 1 1 * * 1.43 ± 0 . 1 8  Values are mean ± S E M between neurons; data from same neurons as table 3.2.11 A ; n = 5, * (relative to control) ** (relative to control and C T Z + K Y N ) P < 0.05, A N O V A test.  Chapter 3. Results I Ran  - 148 -  ratio, and evoked m i n E P S C size decreased as without C T Z and K Y N . A l s o , T T X coapplied with C T Z and K Y N did not affect the amplitude o f pre-stimulation m i n E P S C s . Thus, receptor desensitization or saturation did not mediate T T X effects on quantal size.  3.3.7. Effects of glucose deprivation on STD Because o f the extensive literature relating barbiturate actions to effects on metabolism (see Introduction), the next experiments explored how S T D might be altered during conditions o f limited energy supply, for which brief glucose deprivation was used.  Overall, graded reductions i n glucose concentrations caused a gradual nullification o f S T D . Stepping the glucose concentration to values lower than 25 m M caused a decrease in the amplitude o f E P S C s both at the beginning o f trains and subsequently (Figure 3.32; Table 3.2.12A). This was accompanied by an increase o f the Plateau/Si ratio and a reduction i n the apparent Q N . The post-gap jump was also reduced with decreasing glucose concentrations (Figure 3.32; Table 3.2.12A). Hence, low glucose produced an apparent reduction i n fractional release.  The gradual nullification o f S T D due to glucose deprivations was accompanied by a loss o f the negative covariance terms early i n the train (Figure 3.32 E ) . Figure 3.32E shows that for control conditions i n 25 m M glucose, the covariance term, equal to - 1/N, decreased from cov(Si,S2) to cov(Si,S ); The data suggest that either between-train 5  nonstationarity becomes relatively high, and/or fractional release becomes so small that covariances become nearly undetectable. In principle, the variance is related to p by  Chapter 3. Results I Ran  -149 -  Var(S) = <S>-Q' -(1 - p), which is scarcely affected by p when p is very small, with p = w / N . The covariance, -<Si>-<S2>/N = pQ'<S2> is correspondingly small and therefore readily obscured by between-train non-stationarity, e.g., random failure o f some nerve terminals to participate i n the response.  A s glucose concentrations were stepped down to below 10 m M , the apparent quantal sizes, the variance-mean ratios as well as the size o f evoked m i n E P S C s decreased significantly to values i n the 9-12 p A range (Figure 3.32, 3.33; Table 3.2.12B). The ratio o f quantal contents around the intra-train gap also decreased, with lowering glucose concentrations. Glucose deprivation did not alter the size o f pre-stimulation m i n E P S C s (Figure 3.33; Table 3.2.12B). The effects on apparent quantal size and evoked m i n E P S C size, and lack o f effects on pre-simulation m i n E P S C size, implicate a presynaptic site (or sites), sensitive to glucose deprivation, particularly during periods o f intense synaptic activity, with little or no recovery i n the 20 s between train period.  The effects o f nominally zero glucose conditions were examined on evoked and spontaneous E P S C s . Glucose omission from the perfusion media after 1 m i n resulted i n no change i n the holding current but an irreversible loss o f both spontaneous and evoked EPSCs.  In 3 neurons, large, irreversible increases i n holding current and input  conductance that occurred at 10 min signified a loss o f cell viability. Despite 15-40 m i n periods o f observation i n these neurons, re-establishing control perfusion after the 3 m i n omission did not result i n any recovery o f the E P S C s .  Chapter 3. Results I Ran  -150-  Control (25 mM glucose)  Wash (25 mM glucose)  10 mM glucose  5 mM glucose iWuwrftttfrtr  mmrmmr~  2.5 mM glucose  m 1s 1 o  Contra) • 10 mM glucose £25 mM giucosa)  *  5 mM glucose  nA  •. 2.5 mM glucose  B |  <J  0 04-  •§ 1.0 S 0.03H  Q. £  O  "E 0.02  0.5  trtrttrrtl TrritTTtt 5  10  TO cr  15  3  30  6  10  mW15  stimulus*  0 00  40  I  T:Tirrrn  r  0.01  stimulus #  D  T-»^>  0.03'  '  K0  . 0.02 ^  CO  20-  0:01  to  wH  ff  5  10  15  i=  stimulus #  2  Figure 3.32. Effects o f glucose deprivation on S T D . A ) Current traces o f corticothalamic E P S C s during S T D evoked during brief glucose deprivations. Top: Lowering glucose concentration from 25 to 10 m M (middle trace) decreased the amplitude o f the entire E P S C train without changing the rundown. Middle: Lowering glucose concentration from 25 to 5 m M further decreased the amplitude o f the entire E P S C train and also increased the rundown. Bottom: Reducing glucose from 25 to 2.5 m M abolished S T D (middle trace). B ) Average E P S C s amplitudes (n = 6). C ) Apparent quantal size estimates during glucose deprivation. D ) Quantal content estimates from the neurons i n B and C . E ) Covariance estimates obtained by pairing the 1 E P S C with the 2 - 5 E P S C at different glucose concentrations. The linear regression line is proportional to the rate o f vesicular replenishment. st  n d  t h  Chapter 3. Results I Ran  Table 3.2.12 A : Summary o f parameters o f S T D at different glucose concentrations [glucose] (mM)  25  10  5  2.5  Parameter Si(nA)  1.20± 0.16  0.93 ± 0 . 1 3  0.63 ± 0 . 1 9 * 0.20 ± 0.08*  S (nA)  0.73 ± 0.07  0.42 ± 0.08  0.26 ± 0.06* 0.16 ± 0 . 0 8 *  S2/S1  0.61 ± 0 . 1 1  0.45 ± 0.08  0.42 ± 0.06  0.79 ± 0.09  S12/S10  1.27 ± 0 . 0 8  1.29 ± 0 . 1 3  0.95 ± 0.09  1.04 ± 0 . 0 7 *  Plateau  0.54 ± 0 . 1 6  0.40 ± 0 . 1 9  0.30 ± 0 . 1 1  0.21 ± 0 . 1 3 *  Plateau/Si  0.45 ± 0 . 1 0  0.43 ± 0.09  0.47 ± 0.09  1.05 ± 0 . 1 3 * *  Apparent Q N (nA)  4.31 ± 0 . 3 3  2.69 ± 0 . 3 1 *  1.76 ± 0 . 2 9 * 1.09 ± 0 . 4 1 *  2  (Si 5-20) (nA)  Values are mean ± S E M between neurons; n = 5, * (relative to glucose 25 m M ) ** (relative to all other groups) P < 0.05, A N O V A test. Data are from 10 H z trains.  Chapter 3. Results I Ran  -152 -  Glucose 25 mM  Glucose 5 mM  Glucose 10 mM  Glucose 2.5 mM  Before trains 71 minis In 30 s . a * 10-9 i 4j)  _0.l  u  —  mis'In 30 s  ' 10,914.0  8  I OJ  •  \  6  i  V nL... 2  10  20 30 Size (pA)  40  10.9 .t 4.0  0.0  V  4  0  |  0  10  20 30 Stea (pA)  B After trains 48 minis in6s  i 0  10 ' 20 30 Size(pA)  f  Ds  Cl'1  40  10  20 30 Size (pA)  40  10  20 30 Sizo (pA)  40 10  20  30  40  20 30 Sizs(pA)  40  B-A 20.1 ±4.2  31.2 ±3.4  $  .,.,.... ..^4l-..LU-.J.,_b 0'~{10J • 20: • 130 40 Size (pA) T  I  3  OiZTloj  20 30 Sue (pA)'  40  20 30 'Size (pA)  40  •10  Figure 3.33. Evoked and spontaneous miniature E P S C s during glucose deprivation. A ) Amplitude histograms o f spontaneous m i n E P S C s counted 5 s before the stimulus train (6 repeats, 30 s total) during glucose deprivation produced b y stepping glucose concentration from 25 to 10,5, and to 2.5 m M . B ) Amplitude histograms o f evoked m i n E P S C s counted 1 s after the stimulus train (6 repeats, 6 s total) during the same conditions as i n A . Total m i n E P S C count ('minis') is indicated above histograms. Evoked m i n E P S C sizes were obtained after subtraction o f spontaneous from evoked m i n E P S C histogram (B - A ) . Values next to black arrows are mean size ± S E M . Data are from 1 neuron.  Chapter 3. Results I Ran  - 153 -  Table 3.2.12B: Derived parameters o f S T D at different glucose concentrations [glucose] (mM)  25  10  5  2.5  Parameter cov(Si,S ) (nA ) 2  -0.022 ± 0 . 0 1 3 - 0.009 ± 0.006 0.002 ± 0.003  4xl0"  5  2  Q'(Si)(pA)  35.9 ± 8 . 1  30.1 ± 9 . 1  18.2 ± 1 0 *  10.7 ± 4 . 8 *  Q'(S )(pA)  31.6 ± 7 . 9  29 ± 5 . 1  16.3 ± 3 . 8  9.2 ± 6 . 2 *  Q'(Si5-S ) (PA)  29.2 ± 3 . 8  25.6 ± 4 . 3  12.9 ± 2 . 1 *  8.9 ± 3 . 3 *  OYSJJ-STJV)  0.81 ± 0 . 1 2  0.85 ± 0 . 1 5  28.3 ± 3 . 2  26.7 ± 2 . 5  2  20  0.70  ±0.11  0.83  ±0.14  Q'(Si) Var/Mean (Si5-S o) (PA)  14.6 ± 4 . 1 *  11.4 ± 2 . 1 *  2  Evoked m i n E P S C size (pA)  30.5 ± 3 . 3  Pre-stimulation m i n E P S C size (pA)  11.8 ± 4 . 1  m  x  W15-20  mxilmxo  33 ± 4 19 ± 2  1.23 ± 0 . 0 7  28.1 ± 3 . 4  11.4 ± 2 . 8  26 ± 3 14 ± 3  1.26 ± 0 . 0 8  10.2 ± 2 . 8 **  18.6 ± 2 . 1  10.8 ± 3 . 6  10.2 ± 2 . 9  34 ±  19 ± 8  23  ±  7 8  1.00 ± 0 . 0 5 *  23 ±  7  0.98 ± 0.07*  Values are mean ± S E M between neurons; data from same neurons as table 3.2.12A; n = 5, * (relative to glucose 25 and 10 m M ) ** (relative to all other groups) P < 0.05, A N O V A test.  Chapter 3. Results 1 Ran  -154 -  3.8. Discussion The present studies have demonstrated that pentobarbital produced changes i n quantal parameters during short-term depression o f corticothalamic E P S C s . In control conditions, the quantal aspects o f synaptic transmission was found to behave similarly to that at neuromuscular and calyx o f Held synapses (Scheuss and Neher, 2001; Scheuss et al., 2002). In particular, there were significant negative covariances between responses to the first and second stimuli, as predicted by the binomial/depletion model. In addition, S T D , at different stimulation frequencies, was modulated i n much the same way as at the neuromuscular junction (Elmqvist and Quastel, 1965a). The plateau phase o f E P S C s subsequent to their rundown can be explained by refill eventually matching release characteristics, rather than a different population o f release sites becoming involved. This proposal applies to the stimulation frequencies studied here but may not apply for higher frequencies where refill rates remain unaltered (Wesseling and L o , 2002).  This agreement with the binomial/depletion model provided a validation for using variance/mean ratios, corrected using covariances, to obtain estimates o f apparent quantal size Q' (theoretically Q ( l + C V Q ) ) , at each stimulus. The results then showed that a 2  substantial part o f S T D arises from decline in Q', as at calyx o f Held (Scheuss et al., 2002), another glutamatergic ( A M P A ) synapse, and unlike the neuromuscular junction (Elmqvist and Quastel, 1965a).  The validity o f the statistical estimates o f Q' was also established by their agreement (late in trains) with the size o f m i n E P S C s evoked by the stimulation (cf. Otsu et al., 2004).  Chapter 3. Results I Ran  -155 -  When combined with a pharmacological approach, the statistical method provided estimates for changes i n release parameters during corticothalamic S T D and their modulation by pentobarbital and other drugs or changes i n the bathing medium. A combination o f postsynaptic receptor desensitization and saturation contributed to S T D by reducing the apparent quantal size. Presynaptically, depletion o f readily-releasable quanta mediated the S T D that remained i n the absence o f receptor desensitization and saturation. These findings validate, for the first time, a depletion-based model at a relatively small central synapse and suggest a link between pentobarbital depressant effects and the metabolic requirement during intense processing tasks i n the thalamus.  3.3.8.1. Behaviour of EPSCs in trains Repetitive  stimulation produced  a  frequency-dependent  rundown  of  EPSCs  in  thalamocortical neurons, illustrating a short-term form o f depression, similar to other observations at corticothalamic synapses ( L i et. al. 2003; Reichova and Sherman 2004). The detection o f negative covariances between successive pairs o f E P S C s at the onset o f repetitive stimulation validated the application o f the depletion model, similar to neuromuscular and calyx o f Held synapses (cf. Elmqvist and Quastel, 1965a; Scheuss and Neher, 2001), to obtain estimates o f apparent quantal size at each stage i n the train. The negative covariances were necessary for corrections o f the variance to mean ratios from the initial responses i n a train o f E P S C s , which provided an estimate o f the apparent quantal size. This method also corrected for deviations i n the variance to mean ratio attributable to failure o f axonal conduction and/or action potential invasion into nerve terminals that might occur during prolonged train stimulation.  Chapter 3. Results I Ran  - 156 -  However, the corrected variance-mean model estimated the apparent quantal size and not the "true" quantal size. In other words, this approximation, which is based on assumption o f binomial characteristics, relates to the true quantal size by additional contributions o f within- and between-site variabilities ((1 + C V B ) and (1 + C V w ), respectively; reviewed by Auger and Marty 2000). In other neurons, such variabilities have been estimated by recording at single synapses (Forti et al. 1997) or by comparing distributions o f m i n E P S C s originating at distant terminal branches (Bekkers and Clements 1999). If incorporated into the corrected variance-mean ratio, a future assessment o f within- and between site variability at corticothalamic synapses would approximate the true quantal size alterations during S T D .  Corticothalamic S T D occurred at a frequency range that allows thalamic relay neurons to filter high frequency cortical inputs (cf. Reinker et. al., 2004). A t other C N S synapses that receive high frequency inputs, S T D occurs preferentially at frequencies > 100 H z , whereas, low frequency inputs induce primarily facilitation (von Gersdorff and Borst 2002). Thalamic relay neurons receive a variety o f cortical inputs i n the 2.5-20 H z range that drive or modulate their firing and firing patterns (Reichova and Sherman 2004). Corticothalamic S T D may function as a low-pass filter (Fortune and Rose 2000) and contribute to thalamic processing o f somatosensory inputs particularly during conditions o f intense stimulation and various waking and sleep states.  Chapter 3. Results I Ran  - 157 -  3.3.8.2. Effects of[Ca ] 2+  e  on STD  Corticothalamic S T D was influenced by but did not critically depend on extracellular [ C a ] . In high [Ca ] -containing media, S T D behaved as expected, producing a faster 2+  2+  e  rundown o f E P S C s and higher plateau (cf. Elmqvist and Quastel, 1965a; Scheuss et al., 2002). The heightened plateau o f E P S C s was attributable to an increased quantal content throughout the E P S C train, indicating a presynaptic origin with high [ C a ] increasing 2+  e  P 's. The greater E P S C size subsequent to an intra-train gap also increased i n agreement 0  with raised P . These observations demonstrate a modulatory presynaptic effect o f raised 0  2+  [Ca ] on S T D , closely resembling that seen at neuromuscular junction (Elmqvist and e  Quastel, 1965a).  Surprisingly, reducing [Ca ] did not abolish S T D , disproving the assumption o f a strict e  Ca  dependence. This persistent S T D was not characterized by changes i n quantal  content but rather was attributable to a decrease i n quantal size. This  apparent  dependence o f quantal sizes on [ C a ] might implicate the existence o f a heterogeneous 2+  e  population o f release sites with varying transmitter contents. The concomitant persistence o f S T D and reductions i n quantal size suggest that reducing [ C a ] 2+  e  caused a shift  between populations o f release sites. Alternatively, the smaller quanta could result from a partial release o f transmitter content due to a reduced spread o f the local C a Such effects could provide means to regulate corticothalamic plasticity.  2 +  signals.  Chapter 3. Results I Ran  - 158 -  3.3.8.3. Receptor desensitization and saturation during STD Receptor desensitization and saturation contributed to but did not exclusively mediate corticothalamic S T D , consistent with observations i n calyx o f Held neurons (cf. Scheuss et al. 2002). Pharmacological blockade o f desensitization and saturation slightly reduced but did not abolish the rundown o f corticothalamic E P S C s during train stimulation. The effects demonstrated that S T D is attributable to depletion o f readily-releasable quanta, which is likely a major component.  However, these results imply a paradox. The binomial/depletion model (and negative covariances) indicate that the signals early in the train after the first response are from sites where there was no previous release o f a quantum o f transmitter, especially at frequencies > 10Hz, where refill is relatively small. But those responses show lowered quantal size (Table 3.2.IB; Figure 3.20). A possible explanation is that desensitization and receptor saturation occur because o f overflow o f neurotransmitter from sites where quanta were released to neighbouring sites where quanta are subsequently released (Rossi and Hamann, 1998; Isaacson, 1999; DiGregorio et al., 2002; Telgkamp et al., 2004). This is i n keeping with the observed morphology o f corticothalamic synapses i n the cat (Jones and Powell, 1969).  3.3.8.4. Pentobarbital effects on STD Pentobarbital enhanced S T D by a mechanism that involved a use-dependent decrease i n quantal size, that was not sensitive to blockade o f A M P A receptor desensitization and saturation. The reduction in the apparent quantal size, and evoked m i n E P S C s occurred  Chapter 3. Results I Ran  - 159 -  without effects on ongoing m i n E P S C size observed before stimulation. There are several possibilities which can explain this result. First, the reduction i n quantal size is presynaptic, there being a lowered amount o f transmitter (glutamate) i n each quantum. Second, it could be that with pentobarbital some sites no longer become stimulated presynaptically (e.g. because o f action potential failure) and those that continue to be stimulated are associated with smaller quanta, because o f filtering at distally located dendrites.  Thirdly, it is possible that with stimulation there develops a preferential  release o f pre-existing quanta that have less than normal amounts o f neurotransmitter. Lastly, pentobarbital might enhance or produce a kind o f desensitization not seen normally and not blocked by a combination o f cyclothiazide and kynurenate.  Other studies have demonstrated a preferred action o f pentobarbital to promote the desensitization o f the G l u R 2 subtype o f A M P A receptors (Taverna et al. 1994). However, the actions o f pentobarbital at this receptor subtype expressed i n thalamic neurons (Spreafico et al. 1994), are sensitive to blockade by cyclothiazide (Jackson et al. 2003).  3.3.8.5. Effects of extracellular  alterations on STD  H i g h [ K ] increased whereas low [ K ] reduced the number o f released quanta without +  +  e  producing significant changes  e  i n quantal  size, which indicated a predominantly  presynaptic effect. Unexpectedly, raising [ K ] to 10 m M , while having no effect on the +  e  first E P S C s i n trains, greatly increased the second and subsequent E P S C s (Figure 3.28; Table 3.2.8A, B ) . In other words, facilitation from pulse 1 to pulse 2 was greatly increased. There was also a loss o f the negative covariance between S i and S2, suggesting  Chapter 3. Results I Ran  - 160 -  a large increase i n the rate o f replenishment (refill, a) o f the readily releasable store (Kuromi and Kidokoro, 2004). Increasing [ K ] may have promoted the back propagation +  e  of an action potential which is normally limited by activation o f Ca -gated K 2+  +  channels  subsequent to a local rise in presynaptic [ C a ] . These results imply that local increases i n 2+  [K ] +  e  subsequent to activity at nerve terminals could modify transmitter  release,  transforming a depressed into a facilitated synaptic response (cf. Poolos et al., 1987; Nishimura et al., 1993; Kamiya and Zucker, 1994).  In 10 m M [ K ] , pentobarbital at 200 p M completely blocked the pulse-pulse facilitation +  e  produced by raised [ K ] . This result suggests that whatever the mechanism by which +  e  [ K ] has this effect (note it is opposite to presynaptic depolarization - see Hubbard et al., +  e  1967) it should be sensitive to lower concentrations o f pentobarbital which reduced fractional release without affecting quantal size (cf. Table 3.2.4B). A possible explanation for this is that pentobarbital activated a Ca -gated K channel (Sailer et al., 2004) which 2+  +  blocked the propagation o f an antidromic action potential i n raised [ K ] . This possibility +  e  could be the focus o f further studies.  In low [ K ] , although S T D was apparently unaffected, pentobarbital increased the +  e  amplitude o f the E P S C after the intra-train gap, indicating an increase i n P , or (1 - a), 0  which possibly reflected a selection o f high probability release sites. In summary, altering [K ] +  e  unmasked actions o f pentobarbital on parameters o f quantal release as well as  apparent quantal size.  Chapter 3. Results I Ran  -161 -  3.3.8.6. Effects of tetrodotoxin Because pentobarbital might at least partially act by modifying the presynaptic action potential (Blaustein, 1968), it was worthwhile to study the effect(s) o f tetrodotoxin ( T T X ) , an agent considered to act only by blockade o f voltage-gated N a channels. A t a +  concentration (32 n M ) half that producing block o f E P S C s , T T X equally reduced the apparent quantal size and evoked m i n E P S C size, without affecting pre-stimulation m i n E P S C size (Table 3.2.10B; Figure 3.30). T T X effects presumably resulted from a reduced N a entry into the nerve terminal rather than a decrease i n depolarization per +  action potential, which would reduce effective m's, numbers o f quanta released per stimulus.  3.3.8.7 Effects of lowered glucose In low ambient glucose (5 or 2.5 compared to 25 m M ; Table 3.2.12A, B ) , there were two major effects, lowered quantal size and lowered rundown o f quantal numbers (m's) i n trains (abolition o f S T D in 2.5 m M ) . Reductions in the jump after the omitted stimulus also were consistent with reduced P , compared to controls. This reduction i n P could be 0  G  related to the observation that interference with A T P hydrolysis impairs the transition o f packets from the resting/non-releasable to the release-competent pool (Heidelberger et al. 2002). However, this would amount to a reduction in a (refill o f releasable quanta) rather than reduction i n P . It is therefore likely that energy supply may be critical for both 0  release, per se, and for maintenance o f the readily releasable pool o f packets during periods o f intense stimulation.  Chapter 3. Results I Ran  - 162 -  A s with T T X , quantal sizes did not recover much between trains. Since a specific kind o f desensitization produced by low glucose is hardly likely, there are only two remaining possibilities for the lowered Q' (and evoked m i n E P S C size), namely, reduced amount o f transmitter per quantum and/or selection o f synapses with small quanta (pre- or postsynaptically). O f these possibilities, the first, lowered neurotransmitter per quantum (or packet) seems by far the more likely, since the uptake o f glutamate into synaptic packets critically depends on glucose (Ikemoto et. al., 2002). The present data (Table 3.2.12, A , B ) suggest that glucose deprivation impairs the maintenance o f transmitter content i n the transmitter packets, which constitute the pool or store o f quanta from which outputs are evoked.  General Discussion I Ran  - 163 -  4. General Discussion This thesis provides for the first time, evidence for dual pre- and postsynaptic actions o f the barbiturate, pentobarbital. The most notable actions were to promote oscillogenesis in a thalamic network and to cause short-term plastic modifications o f cortical inputs to thalamic neurons. The details o f these actions have received discussion at the end o f each Results section. Therefore, I w i l l succinctly summarize the most pertinent findings and then, discuss their relevance to a context o f anesthetic mechanisms.  Summary of results Pentobarbital oscillogenesis.  A subanesthetic concentration o f pentobarbital induced  thalamic oscillations i n in vitro preparations. Sustained oscillations at 1-15 H z required electrical stimulation o f the internal capsule, but not elevated temperature or l o w extracellular [ M g ] . 2+  Receptors for glutamate and glycine mediated oscillations i n  ventrobasal nuclei, disconnected from n R T .  Receptors for glutamate and  mediated oscillations i n n R T , disconnected from ventrobasal nuclei.  GABA  Pentobarbital  oscillogenesis occurred i n isolated networks o f the ventrobasal and reticular nuclei, mediated by glutamate receptors with frequency modulation by G A B A - , G A B A - , and A  B  glycine-receptors.  Spermine modulation.  Extracellular spermine acted on the polyamine site o f N M D A  receptors, to increase membrane  rectification on depolarization, to reduce  firing  threshold, and to slow the decay o f corticothalamic EPSPs. The heightened excitability of thalamocortical neurons increased tonic firing evoked by depolarizing current pulses  General Discussion I Ran  -164 -  and E P S P bursts o f action potentials. Spermine increased the rates o f rise and amplitudes of l o w threshold C a  spikes by an unknown mechanism, not mediated by N M D A  receptors. B y increasing the efficacy o f corticothalamic excitation, spermine actions take on importance i n the transformation o f somatosensory signals to tonic and burst discharge patterns during the juvenile stage of rat brain development.  Pentobarbital-spermine  interactions.  Pentobarbital at an anesthetic  concentration  reversed spermine actions that prolonged corticothalamic EPSPs i n thalamocortical neurons. This effect involved postsynaptic interactions at the polyamine site on N M D A receptors. caused  Pentobarbital shortened the E P S P duration and reversed the prolongation  by spermine.  The opposing effects  o f pentobarbital  and  spermine  on  corticothalamic transmission provide a model for anesthetic modulation o f glutamate receptors i n thalamic hyperexcitability.  Pentobarbital  effects on short-term depression (STD).  Pentobarbital enhanced S T D o f  corticothalamic E P S C s by decreasing quantal size i n a use-dependent manner.  These  actions were presumably presynaptic because they were insensitive to pharmacological blockade o f desensitization and saturation o f A M P A receptors. Pentobarbital affected the statistically estimated quantal size (apparent quantal size) and the amplitude o f evoked m i n E P S C s . Prior to electrical stimulation, pentobarbital did not affect the amplitude o f ongoing m i n E P S C s , which reaffirmed a lack o f postsynaptic action on spontaneous minEPSCs.  The effects o f pentobarbital to promote S T D , may have resulted from  General Discussion I Ran  - 165 -  preferred release o f quanta with a small transmitter content,  activation o f a K  +  conductance-mediated shunt o f the presynaptic action potential, or impairment o f evoked release due to blockade o f C a  channels (see below).  Alterations in extracellular [K*]  on short-term depression. H i g h [ K ] increased, +  e  whereas low [ K ] reduced the number o f released quanta. Significant changes i n quantal +  e  size did not accompany these effects, indicating that pentobarbital acted predominantly at presynaptic sites.  Pentobarbital completely blocked the high [K ]-induced facilitation +  early i n a stimulus train. L o w [ K ] by itself did not affect S T D . In combination with +  e  low [ K ] , pentobarbital increased the amplitude o f the E P S C after the intra-train gap, +  e  implicating an increased fractional release due to preferred release from high probability sites.  Effects of tetrodotoxin (TTX) on short-term depression. Application o f T T X at a concentration that was half that producing blockade o f evoked action potentials, reduced the apparent quantal size and evoked m i n E P S C size to the same extent.  T T X did not  affect pre-stimulation m i n E P S C amplitude, suggestive o f a minimal postsynaptic action. The effects o f T T X presumably resulted from a reduced N a entry into the nerve terminal +  and not from a decreased depolarization by each action potential. This interpretation was in agreement with a lack o f effect on numbers o f quanta released per stimulus (effective  m's).  General Discussion I Ran  - 166 -  Glucose deprivation and short-term depression.  Glucose deprivation had two major  effects (Table 3.2.12A, B ) - a reduction in quantal size and lesser rundown o f quantal contents (m's). A ten-fold reduction i n the extracellular glucose concentration abolished S T D and the post-gap jump i n E P S C amplitude. These combined effects were consistent with reduced output probability o f release, P . 0  The combined effects coupled with a  reduced quantal size imply that glucose deprivation impaired the maintenance o f transmitter content i n the packets.  It is plausible that the induced energy shortage  modifies the pool o f quanta from which outputs are normally evoked because glucose deprivation did not affect spontaneous m i n E P S C s .  Initiation ofpentobarbital oscillations Induction o f oscillations by pentobarbital seems paradoxical for a drug usually viewed as a C N S depressant.  Pentobarbital hyperpolarizes thalamocortical neurons, acting by  multiple mechanisms over a subanesthetic concentration range (Wan and Puil, 2002; W a n et al., 2003). Hyperpolarizing activation and deinactivation o f intrinsic currents would promote oscillogenesis i n a thalamic slice network (cf. M c C o r m i c k and Pape, 1990), modulated by receptor-gated currents (cf. Steriade et al., 1997).  In the present study, G A B A , G A B A , and glycine receptors modulated, but none was A  B  essential for pentobarbital-induced oscillations (cf. Table 4.1). Glycine receptors likely were critical for oscillations i n a dorsal thalamic network, divested o f G A B A e r g i c inhibition. Glycine is not a recognized neurotransmitter i n the thalamus, where G A B A  General Discussion 1 Ran  - 167 -  Table 4.1: Receptor involvement i n pentobarbital-induced oscillations  Recording Site  Ventrobasal nuclei  Ventrobasal nuclei, without n R T  nRT  n R T , without ventrobasal nuclei - not tested  Activation of Receptors for  Glutamate  GABA  Glycine  Essential  Modulate frequency  Modulate frequency  Essential  No apparent role  Essential  Essential  No apparent role  Essential  No apparent role  General Discussion I Ran  - 168 -  mediates transmission by reticulothalamic and some interneuronal pathways (Steriade et al., 1997). The thalamus exhibits strychnine-binding sites, m R N A for glycine receptor subunits and expresses a subunit protein (Zarbin et al., 1981; Malosio et al., 1991; Naas et al., 1991). Conceivably, activation o f glycine  receptors  may  mediate  synaptic  inhibition i n ventrobasal nuclei o f rat (cf. Tebecis, 1974; Ghavanini et al., 2005).  A subanesthetic concentration o f pentobarbital induced oscillations i n a corticothalamic network. These oscillations required glutamatergic excitation, but not  GABAergic  inhibition from n R T . Glycine receptors were essential for the oscillations i n ventrobasal nuclei, isolated from n R T . G A B A receptors were essential for the oscillations i n n R T , isolated from ventrobasal nuclei. Thus, pentobarbital can induce oscillogenesis i n either ventrobasal nuclei or nRT, independent o f their reciprocal synaptic connectivity.  The pentobarbital-induced oscillations i n slices may have some relevance to the genesis o f spindling i n the E E G during barbiturate administration (see Introduction). The stimulation may have mimicked the slow rhythm that neocortex imposes through glutamatergic pathways  (Steriade et al., 1991, 1997; von Krosigk et al., 1993). The  insensitivity to temperature was possibly due to compromised network behavior i n the slice (cf. Andersen and Andersen, 1968). The sustained thalamocortical oscillations observed i n the slice network may reflect pentobarbital actions that produce sedativehypnotic effects in vivo (Nelson et al., 2002). Substances that mimic this oscillogenesis may have potential as sedative-hypnotic drugs. The stationary oscillations may represent a model o f sedation-hypnosis, amenable to pharmacological analysis.  General Discussion I Ran  Pentobarbital likely acted on M g  - 169 -  independent sites. In either normal or low external  [ M g ], an anesthetic concentration (200 u M ; cf. Sato et al., 1995) first increased and then abolished oscillations. Jacobsen et al., (2001) observed that at lower concentrations (100 \\M) pentobarbital eliminated ventrobasal oscillations i n low [ M g ] . Disappearance 2+  o f oscillations may have resulted from a G A B A  A  receptor shunt o f glutamate excitation  (Wan et al., 2003 and Sawada and Yamamoto, 1985). The new and previous results are consistent with the marked C N S depression that occurs during deep anesthesia in vivo (Satoetal., 1995).  Spermine modulation of thalamic excitability  The endogenous polyamine spermine applied i n the 1-200 pJVl range enhanced the excitability o f thalamocortical neurons i n specific ways that were consistent with a neuromodulator role at P12-P15 stage o f development.  Spermine actions on N M D A  receptors produced a heightened state o f excitability which were viewed as prolonged E P S P s , and increased bursting and tonic firing o f action potentials. To a large extent, these effects resulted from increased membrane rectification on depolarization and a reduced threshold for action potential genesis. Spermine also modulated the burst mode by increasing the rate o f rise and amplitude o f low threshold C a  2 +  firing  spikes (LTSs).  This unusual effect did not involve interaction with glutamate receptors. The modulation o f corticothalamic excitation and L T S s o f M G B neurons may be critical i n the transformation o f auditory signals i n gerbil thalamus at the P14 stage o f development.  General Discussion I Ran  -170 -  Spermine is widely distributed i n rodent and human brain (Harman and Shaw, 1981; Morrison et al., 1995). A membrane transporter appears to maintain low extracellular concentrations o f <1 f i M (Dot et al., 2000).  These concentrations may increase on  N M D A stimulation to > 50 f i M i n striatal neurons o f adult rat brain (Fage et al., 1992). The effects o f spermine on N M D A receptors and low threshold C a  2 +  spikes i n juvenile  M G B neurons (ED o= ~100 | i M ) are consistent with neuromodulatory actions at high 5  micromolar concentrations (Williams, 1997). Given the role o f N M D A receptors during development, such modulation by spermine is likely important for learning processes ( C h i d a e t a l . , 1992).  The present results are relevant to the normal function o f the central auditory system. The N M D A receptor-mediated effects o f spermine would enhance the ability o f thalamic neurons to detect simultaneous inputs, as i n coincidence detection. For example, an overexpression o f the spermine-sensitive N R 2 B subunit (Williams et al., 1994) prolongs E P S P s and shortens the time window between two coincident signals i n hippocampal neurons (Tang et al., 1999). In thalamic neurons, the generation o f synchronous activity may  involve coincidence detection (Roy and Alloway, 2001) as well as amplitude  selectivity i n the M G B neurons (Kuwabara and Suga, 1993).  The effects o f spermine on the L T S s o f M G B neurons may have relevance for conscious or sleep states and disorders o f consciousness. The L T S is essential i n the generation o f  General Discussion IRan  -171-  bursting and oscillatory activity in the auditory nuclei (Hu, 1995; Tennigkeit et al., 1996). B y increasing the rate o f rise and amplitude o f the L T S and slowing its decay, spermine modulation may increase an M G B neuron's responsiveness o f neurons at hyperpolarized potentials (Hu et al., 1994) to inputs during these states (He and H u , 2002). Modulation by spermine may have importance for bursting behavior during sleep states whereas excessive modulation may occur i n absence epilepsy as i n audiogenic seizures (Porta et al., 1981), sensitive to blockade by polyamine antagonists (Kotlinska and Liljequist, 1996).  Pentobarbital reversal of spermine modulation Pentobarbital, at an anesthetic concentration, modulated corticothalamic transmission by postsynaptic actions at the polyamine site on N M D A receptors. Pentobarbital effects included shortening o f the duration o f EPSPs and reversal o f their prolongation by extracellular spermine. Consistent with pentobarbital shortening o f burst durations and mean open times o f N M D A - m e d i a t e d single channel currents (Charlesworth et al., 1995), these actions on N M D A - m e d i a t e d transmission may contribute to its depressant effects on oscillogenesis.  L i k e other polyamines, spermine increases the potency o f barbiturates to induce general anesthesia (Daniell, 1992). The basis for this enhancement is presently unclear because of the observed opposing actions o f pentobarbital and spermine on the prolongation o f N M D A mediated EPSPs. Indeed, pentobarbital depression o f transmission prevented the  General Discussion I Ran  - 172 -  prolongation o f N M D A responses caused by spermine. The effects and interactions o f pentobarbital might have relevance for various forms o f epilepsy. For example, N M D A receptors are activated i n thalamocortical neurons during the development o f spike-andwave discharges i n a strain o f genetic absence epilepsy rats (Koerner et al., 1996). Similarly, an injection o f N M D A into subthalamic nuclei o f rats generates audiogenic seizure behaviour (Faingold el al. 1989) and increases neuronal firing i n M G B neurons (N'Gouemo  and  Faingold,  1997).  Hence,  pentobarbital  actions  that  shorten  corticothalamic E P S P s might alleviate N M D A mediated epileptic seizures.  Presumed presynaptic actions ofpentobarbital A n anesthetic concentration o f pentobarbital enhanced S T D at corticothalamic synapses. The increased S T D can be attributed to a presynaptic use-dependent action that produced small  size quanta.  The effects  persisted  during blockade o f A M P A  receptor  desensitization and saturation and were not accompanied by alterations i n spontaneous m i n E P S C size. The rapid fall i n quantal size implies a rather small population o f preformed quanta. This was evident i n the fast reduction i n quantal size after the 3  r d  stimulus i n the train. The reduction can also be explained by use-dependent inactivation of C a  2 +  channels which reduces C a  2 +  entry into terminals early i n the train and impairs  the formation o f releasable quanta. Pentobarbital, at a similar concentration, reduces the amplitude o f miniature EPSPs by impairing C a al., 2003).  B y reducing C a  2 +  2 +  entry into nerve terminals (Baudoux et.  entry into corticothalamic terminals, pentobarbital could  have interfered with glutamate release by selectively affecting a population o f small size and/or partially refilled quanta.  General Discussion I Ran  - 173 -  Similar effects of tetrodotoxin and pentobarbital Tetrodotoxin mimicked the pentobarbital enhancement o f short-term depression. The effects o f T T X to reduce apparent quantal size (Q') i n a use-contingent manner differed from that o f pentobarbital in that recovery from T T X between iterated trains was incomplete (Table 3.2.10B). T T X had a progressive effect between trains, reducing the number o f participating release sites.  The same interpretations o f a lowered Q ' (see  Section 3.3.8.4., page 159), apply for T T X as for pentobarbital, except that a specific kind o f TTX-induced desensitization seems implausible. The effects o f T T X on Q ' occurred at half the concentration which blocked action potentials, and likely resulted from reduced Na  +  entry into the nerve terminal, rather than from blockade o f depolarization. The  similar amplitude o f pre-stimulation and evoked m i n E P S C s observed here during T T X application suggests a close resemblance to m i n E P S C s , or true m i n E P S C s , observed i n other studies during complete blockade o f action potentials at T T X concentrations > 1 p M (cf. Edwards et al., 1990).  Given the high selectivity o f T T X , these results may imply that these drugs modulate Na -dependent processes that pump glutamate into releasable packets at corticothalamic +  terminals. There was a large difference i n the concentrations o f T T X and pentobarbital that affected transmitter release. Unlike pentobarbital, T T X would likely block axonal invasion o f the presynaptic action potential. In axons, such blockade by pentobarbital is evident only at millimolar concentrations (Blaustein, 1968). Pentobarbital depression o f thalamic firing occurs at much lower, subanesthetic doses and is attributable to a K  +  conductance shunt (Wan et al., 2003). Hence, pentobarbital i n the present experiments  General Discussion I Ran  -174 -  could have shunted the presynaptic action potential by activating K conductances i n the +  nerve terminal.  Effects of glucose deprivation on short-term depression The similar effects  o f glucose deprivation imply that enhancement  o f short-term  depression by pentobarbital was, in part, attributable to its depressant actions on metabolism (see Introduction). The reduction in fractional release during glucose deprivation could be explained by a drop i n the A T P content, which impairs the maintenance o f a release-competent pool o f transmitter vesicles (Heidelberger et al. 2002). B y reducing the energy supply, pentobarbital may impair both release, per se, and the maintenance o f the readily releasable pool o f transmitter packets, particularly during periods o f increased activity-dependent release o f transmitter.  Apparent redundancy in pentobarbital effects on transmitter release The mimicry o f action potential blockade and energy deprivation as well as multiple actions on quantal release (cf. W a n et al., 2003), suggests that pentobarbital may have reduced the size o f the store and transmitter content o f quantal packets. Pentobarbital inhibits glycolysis which provides most o f the A T P at synaptic terminals (Crane et al., 1978). A decrease i n the A T P content at nerve terminals impairs processes that maintain the content (Dcemoto et al., 2003) and availability (Heidelberger et al. 2002) o f readilyreleasable quanta. It is also possible that conditions o f glucose deprivation or blockade o f Na  +  entry by T T X affected separate processes, each impaired by high micromolar  concentrations o f pentobarbital.  General Discussion I Ran  - 175 -  The effects o f pentobarbital, T T X , and energy deprivation on transmitter release have an alternative explanation.  These effects may involve impairment o f action potentials at  specific sites, sparing distal sites o f transmitter release. Selective blockade would result i n a preferred release o f the remaining, highly releasable small quanta, and reduced quantal size as observed postsynaptically.  This process is compatible with  the  observations that pentobarbital depletes docked packets at synaptic active zones (Jones and Devon, 1978; Hajos et al. 1978).  Other studies have suggested that heterogeneity in contents o f transmitter packets contributes to variation i n quantal amplitude (Edwards, 1990).  This proposal is  tantamount to the proposition that release o f partially filled quanta occurs normally. The lowered quantal size due to incomplete filling i n the low glucose condition would then be seen as an exaggeration o f something that also occurs normally. However, it is also possible that small quanta are preferentially released when energy supply is l o w and the same could be true in the presence o f T T X and pentobarbital.  Mechanisms ofpentobarbital enhancement of short-term depression There are at least four distinct mechanisms that could explain the ability o f pentobarbital to reduce quantal size: 1) a selective process whereby pentobarbital depleted large size quanta, sparing the smaller size quanta. Ca  2 +  For example, pentobarbital can interfere with  entry (Charlesworth et al., 1995) into active zones critical for transmitter release at  nerve terminals. The selective process is consistent with known actions o f pentobarbital that deplete a subset o f docked packets i n active zones (Jones and Devon, 1978; Hajos et  General Discussion I Ran  - 176 -  al. 1978); 2) a pentobarbital induced suppression o f glycolysis and associated A T P - and Na  +  -dependent  glutamate uptake (see Introduction) which may reduce  transmitter  content per packet (cf. Gueldry et. al. 1987; Ikemoto et al. 2002); 3) a pentobarbital interference with C a  2 +  entry (Charlesworth et al., 1995) that alters the kiss-and-run  (Pocock and Richards, 1987; A n and Zenisek, 2004) or porocytotic (Kriebel et al., 2001) type openings o f fusion pores.  This blockade would limit the amount o f released  transmitter; and, 4) a pentobarbital-evoked desensitization o f postsynaptic cyclothiazideinsensitive receptors.  This desensitization would reduce the postsynaptically observed  quantal size, i n response to a given amount o f transmitter (see Introduction). A t present, there is no evidence for such receptors on thalamic neurons (cf. Chen et. al. 2002).  In principle, there are many known pre- and postsynaptic sites or targets for barbiturate action that could be involved i n these experiments on corticothalamic S T D (Figure 4.1). For example, one might expect pentobarbital to activate a K  conductance-mediated  +  shunt o f presynaptic action potentials (Wan et al., 2003), limiting the activation o f voltage-gated C a  channels and therefore release probability (P ). A l s o a priori, one 0  might expect that, by interfering with metabolism, pentobarbital might inhibit the transfer of resting packets to the releasable store (Heidelberger et al. 2002).  The  present results, however, contradict these expectations, particularly those  from  experiments with raised [ K ] . In raised [ K ] , pentobarbital completely inhibited a novel +  +  e  e  action o f [ K ] to increase quantal release only after the first stimulus i n the train. This +  e  finding suggests that a major effect o f pentobarbital at lower concentrations might be to  General Discussion I Ran  -177 -  suppress normal modulation o f transmitter release by changes i n local [ K ] at synapses, +  e  produced by activity at nearby synapses.  A n elevated [ K ] would arise from release +  e  sites/boutons that have not released quanta after the first stimulus. Then, it follows from the depletion model that the transmitter released from nearby synapses would have contributed to the observed greater responses to the second stimuli i n a raised K  +  condition (Figure 3.28 A ; Table 3.2.8A). The inferred 'cross-talk' between neighbouring synapses, and its modification by drugs, represent an interesting area for further study.  Limitations The methods and experimental approaches i n this thesis possess a number o f limitations. The in vitro oscillations induced by pentobarbital measurement  o f the intracellular changes  did not include concomitant  in electrical membrane  properties  that  participate i n thalamocortical oscillogenesis (von Krosigk et al., 1993). Simultaneous intra- and extracellular recording during pentobarbital application and oscillogenesis would enable identification o f ionic conductances that are essential for producing synchronous rhythmic activity.  The study o f extracellular application o f spermine on excitability did not exclude possible intracellular interactions.  Spermine may have gained access to the cytoplasm through  membrane transporters (Fage et al., 1992; Dot et a l , 2000). For example, an intracellular action o f spermine occurs at Ca -permeable A M P A receptors and inward rectifier K 2+  channels (Williams, 1997), which boosts thalamic excitability.  +  Unlike inward rectifier  General Discussion I Ran  K  - 178 -  channels, however, C a -permeable A M P A receptors have not been identified i n the  thalamus.  The present study involved stimulation o f multiple converging corticothalamic fibres which could result i n conduction failure at axonal branches, especially late i n the stimulus train. Future studies using focal stimulation (Dunant and Muller, 1986; Quastel and Saint, 1988) may attempt to minimize conduction failure which also may be assessed by simultaneous or independent extracellular recording o f fibre volleys (Poolos et al., 1987).  The binomial model is limited by an assumption o f independence between release sites (cf. Vere-Jones, 1966). Not surprisingly, many studies have not reported the existence o f negative covariances. The lack o f negative covariances i n these studies may be explained by a positive correlation between active release sites which nullifies the negative covariance that would occur early i n the train. Hence, a high level o f branch conduction failure may explain the lack o f negative covariances i n other studies.  The negative  covariances, here, implicates a minimal contribution o f conduction failure to the data.  In the present thesis, the variance and covariance were obtained from sequential repeats o f responses.  This procedure minimized slow trends that developed between trains  throughout the experiment.  O n the other hand, this approach prevented assessment o f  slow changes due to drug effects.  The procedure may have resulted i n an increased  sampling error or caused an underestimation o f quantal parameters.  General Discussion I Ran  - 179 -  The identification o f m i n E P S C size was difficult to estimate within a train and hence, restricted to pre- and post-train periods. The difficulty is attributable to the low signal-tonoise ratio o f intra-train m i n E P S C s . A method that would correct for the time course o f the decay o f E P S C may enable accurate detection o f intra-train m i n E P S C s . The b i n size i n the present study resulted i n a signal resolution with a minimum value o f 5 p A . Studies that have an improved signal-to-noise ratio may provide information on smaller changes i n quantal size. This information then could be combined with rise times for estimating the distance between release sites and the synapse.  Unequivocal demonstration o f a drug effect requires observations o f full reversibility. In most cases, the drug effects were reversible. However, T T X application at concentrations > 64 n M or the omission o f glucose from the A C S F , produced effects that were irreversible during the period o f observation, followed by loss o f neuronal viability. A n inability to observe excitation did not allow a more exact determination o f pentobarbital's effects on spontaneous m i n E P S C s , normally studied i n the presence o f > 1 | j M [ T T X ] . Future studies that incorporate techniques for differentiating evoked and spontaneous transmitter release (cf. X u and Sastry, 2003), may address this issue. Extensive studies are required to examine reversible effects o f glucose deprivations over a much wider concentration range than used here. The new studies may facilitate a distinction between pentobarbital effects on spontaneous and evoked release, for an assessment o f its longterm effects on transmission.  General Discussion I Ran  -180-  Conclusion The  present  studies  have  pentobarbital  actions  on  provided several thalamic  neurons.  new  contributions  to  understanding  A  subanesthetic  concentration  of  pentobarbital induced network oscillations in vitro, which would modulate thalamic inhibition and facilitate corticothalamic excitation in vivo. Another original contribution is the validation o f the binomial depletion model o f transmitter release at a conventional C N S synapse. The analysis has led to novel interpretations about pre- and postsynaptic anesthetic  actions  of  pentobarbital  corticothalamic excitation.  on  short-term  plasticity  during  repetitive  These studies also showed interesting similarities i n its  actions that reduced quantal size, to imposed conditions o f impaired N a entry into nerve +  terminals and energy shortage due to glucose deprivation.  It seems significant that  pentobarbital reversed the facilitating effect o f elevated [ K ] which typically promotes a +  e  plastic change i n transmission. This action represents a new type o f synaptic modulation by barbiturate, complementing known anesthetic actions on thalamic neurons. M a n y o f the actions o f pentobarbital, including actions on quantal parameters, are summarized i n the schematic diagram o f Figure 4.1. The method and analysis technique described for pentobarbital actions in this thesis provide a model for examining the pre- and postsynaptic effects o f drugs on transmission i n the C N S .  General Discussion I Ran  - 181 -  Na* channel Kr-s*  j  channel  P <-~  SL  . / " .  fa)  '"•  ^  /JC*  X  = > s\ j \  ^channel  fa  •fe.  \ ^ \ . > * w ATP 4"--> Glucose — T  ( glut  ^  Reduced pore opening^ AMPA receptors » *  /  ^ Glucose n  n  \  ADP+P,  / " \  / I <**  A  .  ~ Pentobarbital  Vesicular selection.. / / / .-a.-! ...-^ ,,A  ./ if  _  fa,  H  *  ^  ^I"  \A Ca' channel -  ' ~~"  "  ^  ATP  h  i  n  n  -St.  ^-  Na* glu  NMDA receptors  Pentobarbital Figure 4.1. Possible synaptic targets for pentobarbital actions during corticothalamic S T D . Pentobarbital enhancement o f S T D might involve ion channel modulation, inhibition o f Na -dependent glutamate uptake into readily releasable packets, suppression o f glycolytic A T P , and impairment o f glutamate exocytosis. Actions on ion channels include: induction o f K channel-mediated shunt, blockade o f voltage-gated C a channels, blockade o f N a channel-mediated action potentials. Pentobarbital might also inhibit carrier-mediated glucose and N a transport into the terminal cytoplasm. Pentobarbital suppression o f glycolysis could reduce ATP-dependent uptake o f glutamate into packets. In the case o f a heterogenous population o f packets, penotbarbital's inhibition o f voltage gated C a channels might select for small size quanta. Pentobarbital may also shorten the formation o f fusion pores thereby reducing exocytotic release o f glutamate. Postsynaptically, pentobarbital could promote desensitization o f A M P A or N M D A receptors. +  +  2 +  +  +  2 +  -182 -  Bibliography 1 Ran  Bibliography Aldridge W N , Parker V H . Barbiturates and oxidative phosphorylation. Biochem J 1960, 76:47-56. Aimers W , Neher E . 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