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Supraspinal actions of pentobarbital on transmission through the spinothalamic tract Namjoshi, Dhananjay 2007

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SUPRASPINAL ACTIONS OF PENTOBARBITAL ON TRANSMISSION THROUGH THE SPINOTHALAMIC TRACT by Dhananjay Namjoshi M. Pharm, University of Mumbai, 2003 B. Pharm, University of Mumbai, 1999  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in The Faculty of Graduate Studies (Pharmaceutical Sciences)  THE UNIVERSITY OF BRITISH COLUMBIA December 2007 © Dhananjay Namjoshi, 2007  ABSTRACT  Despite the advances made in our understanding of the molecular mechanistic actions of general anesthetics very little is known about the in vivo neural circuits involved in creating the state of general anesthesia. To date the common consensus is that general anesthetics act ubiquitously within the CNS. Recently, (Devor and Zalkind, 2001) have reported that microinjections of pentobarbital (PB) into a discrete brainstem focal area of conscious rats induced a classical, reversible general anesthesia‐like behavioral state. The authors concluded that this area, termed the mesopontine tegmental anesthesia area (MPTA), may be important for the induction of general anesthesia. The purpose of the present project was to study the neurophysiological basis of the analgesia, which accompanied the state of general anesthesia induced by PB microinjections into the MPTA that was reported by (Devor and Zalkind, 2001). Here, sensory inflow via the spinothalamic tract (STT), a classical spinal nociceptive pathway in the rat, was assessed using single neuron extracellular recording techniques before, during and after microinjections of PB into the MPTA.  Spontaneous firing rate (SFR), antidromic firing index (FI) and sciatic as well as sural nerve‐evoked responses (Sc‐, Su‐ER) of STT neurons in isoflurane‐anesthetized rats were quantified before as well as 2, 15, 30 and 60 min following bilateral microinjections of either PB (200 μg/side) or vehicle control solution (Vh, 1 μL/side) into the MPTA.  The group mean SFR, FI as well as magnitudes of Sc‐, Su‐ER of STT neurons were significantly  and  reversibly  reduced  following  PB  microinjections  compared  to  ii  corresponding baseline measurements. There were no significant changes in any of the three parameters following microinjections of Vh compared to the pre‐microinjection baseline responses.  The results from this study indicate that analgesia, which occurs during the anesthesia‐like state following microinjections of PB into the MPTA, may be due to attenuation of sensory inflow through the STT. The suppression of STT neurons likely occurs via direct and/or indirect descending pathways from the MPTA to the spinal cord. This study provides the first direct electrophysiological evidence for the analgesia caused by PB microinjections into the rat MPTA.  iii  TABLE OF CONTENTS ABSTRACT......................................................................................................................................... ii TABLE OF CONTENTS .................................................................................................................. iv LIST OF TABLES............................................................................................................................viii LIST OF FIGURES ............................................................................................................................. x LIST OF SYMBOLS AND ABBREVIATIONS ...........................................................................xv ACKNOWLEDGEMENTS.............................................................................................................xix DEDICATIONS ...............................................................................................................................xxi CHAPTER 1: GENERAL INTRODUCTION................................................................................. 1 1.1. Mechanism(s) of Actions of General Anesthetics: From Lipid to Protein Theory...... 2 1.2. Central Sites of General Anesthetic Actions...................................................................... 3 1.3. Mesopontine Tegmental Anesthesia Area: Potential Central Site for the Induction of General Anesthesia .......................................................................................................... 4 1.4. The Spinothalamic Tract ........................................................................................................ 9 1.5. Study Rationale...................................................................................................................... 10 1.6. Research Hypothesis............................................................................................................. 12 1.7. Research Objectives .............................................................................................................. 12 CHAPTER 2: MATERIALS AND METHODS............................................................................ 16 2.1. SURGICAL PROCEDURES ................................................................................................ 16 2.1.1. Anesthetic Induction......................................................................................................... 16 2.1.2. Tracheotomy ....................................................................................................................... 18 2.1.3. Sciatic and Sural Nerve Surgery ..................................................................................... 19 2.1.4. Thoracolumbar Laminectomy ......................................................................................... 19 2.1.5. Craniotomy.......................................................................................................................... 20  iv  2.1.6. T13‐L1 Vertebral Immobilization Procedures ................................................................ 21 2.2. EXTRACELLULAR RECORDING PROCEDURES ........................................................ 22 2.2.1. Antidromic Identification of Spinothalamic Tract (STT) Neurons ......................... 22 2.2.2. Electroencephalogram....................................................................................................... 24 2.2.3. Spike Amplification, Recording and Data Acquisition Procedures ........................ 25 2.2.4. Baseline Electrophysiological Recording Procedures................................................. 26 2.2.4.1. Spontaneous Spike Activity .................................................................................. 26 2.2.4.2. Antidromic Firing Index......................................................................................... 27 2.2.4.3. Peripheral Nerve Stimulation‐Evoked STT Responses ................................... 28 2.3. DRUG AND CONTROL SOLUTIONS ............................................................................ 29 2.4. INTRACEREBRAL MICROINJECTIONS PROCEDURE............................................. 30 2.5. DRUG/VEHICLE CONTROL‐RESPONSE STUDIES ................................................... 32 2.6. BRAIN PERFUSION AND HISTOLOGY ........................................................................ 34 CHAPTER 3: RESULTS ................................................................................................................... 39 3.1. SPINOTHALAMIC TRACT NEURONS: GENERAL CHARACTERISTICS............ 40 3.2. STUDIES OF THE ELECTROPHYSIOLOGICAL PARAMETERS ............................. 44 3.2.1. Three Groups of Spinothalamic Tract Neurons According to the Treatment Protocol............................................................................................................................... 45 3.2.2. ELECTROPHYSIOLOGICAL PARAMETERS AT THE BASELINE ....................... 48 3.2.2.1. Spontaneous Firing Rate ........................................................................................ 48 3.2.2.2. Antidromic Firing Index......................................................................................... 49 3.2.2.3. Peripheral Nerve‐Evoked STT Responses.......................................................... 49 3.2.2.3.1. Sural Nerve‐Evoked STT Responses ............................................................ 51 3.2.2.3.2. Sciatic Nerve‐Evoked STT Responses .......................................................... 55  v  3.2.3. EFFECTS OF BILATERAL MICROINJECTIONS OF VEHICLE CONTROL SOLUTION/PENTOBARBITAL INTO THE RAT MPTA ON THE ELECTROPHYSIOLOGICAL PARAMETERS OF STT NEURONS...................... 59 3.2.3.1. MICROINJECTIONS OF VEHICLE CONTROL SOLUTION ....................... 59 3.2.3.1.1. Spontaneous Firing Rate: Vehicle Control Microinjections .................... 59 3.2.3.1.2. Interspike Interval Data: Vehicle Control Microinjections...................... 64 3.2.3.1.3. Antidromic Firing Index: Vehicle Control Microinjections..................... 67 3.2.3.1.4. Sural Nerve‐Evoked STT Responses: Vehicle Control Microinjections ................................................................... 70 3.2.3.1.5. Sciatic Nerve‐Evoked STT Responses: Vehicle Control Microinjections ................................................................... 74 3.2.3.2. MICROINJECTIONS OF PENTOBARBITAL................................................... 82 3.2.3.2.1. Spontaneous Firing Rate: Pentobarbital Microinjections......................... 82 3.2.3.2.2. Interspike Interval Data: Pentobarbital Microinjections.......................... 87 3.2.3.2.3. Antidromic Firing Index: Pentobarbital Microinjections ......................... 90 3.2.3.2.4. Sural Nerve‐Evoked STT Responses: Pentobarbital Microinjections ....................................................................... 94 3.2.3.2.5. Sciatic Nerve‐Evoked STT Responses: Pentobarbital Microinjections ....................................................................... 98 3.3. RESULTS OF HISTOLOGY OF MICROINJECTION SITES ..................................... 106 CHAPTER 4: DISCUSSION ......................................................................................................... 110 4.1. Introductory Remarks......................................................................................................... 110 4.2. Technical Considerations: Animal Model, Study Design, Histology, and Statistical Analysis ...................................................................................................... 112 4.3. STT Neurons: General Properties .................................................................................... 119  vi  4.4. Inhibition of Spontaneous and Evoked Responses of STT Neurons by Pentobarbital Microinjections into the MPTA ............................................................ 122 4.4.1. Spontaneous Firing Rate ................................................................................................ 123 4.4.2. Interspike Interval Parameters...................................................................................... 128 4.4.3. Antidromic Firing Index................................................................................................. 129 4.4.4. Peripheral Nerve‐Evoked STT Responses .................................................................. 132 4.5. Time Course of Effects of Pentobarbital on the Spike Activity of STT Neurons ... 136 4.6. Possible Neural Mechanism(s) of Pentobarbital‐Mediated Suppression of STT Neurons through the MPTA............................................................................................ 137 4.7. MPTA: A Possible Pronociceptive Center in the Brain? .............................................. 147 4.8. Future Directions ................................................................................................................. 150 CHAPTER 5: SUMMARY AND CONCLUSIONS .................................................................. 155 REFERENCES.................................................................................................................................. 157 APPENDICES.................................................................................................................................. 179 Appendix A.................................................................................................................................. 179 Appendix B .................................................................................................................................. 180  vii  LIST OF TABLES  Table 3.1  Summary of effects of bilateral microinjections of vehicle control solution into the MPTA of isoflurane‐anesthetized rat preparation on interspike interval parameters of STT neurons ……………………….…....66  Table 3.2  Summary of effects of bilateral microinjections of vehicle control solution into the MPTA of isoflurane‐anesthetized rat preparation on the electrophysiological parameters of Group I STT neurons.……….…....79  Table 3.3  Summary of effects of bilateral microinjections of vehicle control solution into the MPTA of isoflurane‐anesthetized rat preparation on the electrophysiological parameters of Group III STT neurons…...…...…80  Table 3.4  Summary of effects of bilateral microinjections of vehicle control solution into the MPTA of isoflurane‐anesthetized rat preparation on electrophysiological parameters obtained by combining the results obtained from Groups I and III STT neurons…………………………………...81  Table 3.5  Summary of effects of bilateral microinjections of pentobarbital into the MPTA of isoflurane‐anesthetized rat preparation on interspike interval parameters of STT neurons…...…………………………...……………89  Table 3.6  Summary of effects of bilateral microinjections of pentobarbital into the MPTA of the isoflurane‐anesthetized rat preparation on the electrophysiological parameters of Group II STT neurons………………103  viii  Table 3.7  Summary of effects of bilateral microinjections of pentobarbital into the MPTA of isoflurane‐anesthetized rat preparation on the electrophysiological parameters of Group III STT neurons …........................104  Table 3.8  Summary of combined results of effects of bilateral microinjections of pentobarbital into the MPTA of the isoflurane‐anesthetized rat preparation on electrophysiological parameters of Group II and Group III STT neurons…………………..…………………………….…………105  ix  LIST OF FIGURES  Figure 2.1  Schematic of the experimental setup for extracellular recording of STT neurons and intracerebral microinjections of pentobarbital and/or vehicle control solutions into the MPTA…...…………………………...36  Figure 2.2  Photograph of microinjection cannula system used for intracerebral microinjections of pentobarbital/vehicle control solutions……………………37  Figure 2.3  Flowchart of the steps involved in electrophysiological parameter recording and microinjections procedures……………………………………..38  Figure 3.1  Criteria used for antidromic identification of STT neuron……………………42  Figure 3.2  Schematic diagram of estimated thalamic stimulation and spinal recording sites of STT neurons recorded in isoflurane‐anesthetized rat preparation……………………………………………………………………..43  Figure 3.3  Correlation between axonal conduction velocity and spinal recording depth of STT neurons….………………………………………………….……….46  Figure 3.4  Schematic explanation of STT neurons classified into three groups based on the treatment(s) they received………………………………………...47  Figure 3.5  Correlation of baseline spontaneous firing rate with spinal and axonal conduction velocity of STT neurons……………………………………………..50  Figure 3.6  Example of sural nerve‐evoked STT responses……………...…………………53  x  Figure 3.7  Absence of habituation of sural nerve‐evoked STT responses......……………54  Figure 3.8  Example of sciatic nerve‐evoked STT responses……………….………………57  Figure 3.9  Absence of habituation of sciatic nerve‐evoked STT responses.……………...58  Figure 3.10  Effect of bilateral microinjections of vehicle control solution into the MPTA of isoflurane‐anesthetized rat preparation on the spontaneous rate of firing STT neurons.....…………..………………………...….……………61  Figure 3.11  Spontaneous firing rate of each STT neuron before and after bilateral microinjections of vehicle control solution into the MPTA of isoflurane‐ anesthetized rat preparation..…………………………………….………….…...62  Figure 3.12  Example of a continuous ratemeter histogram trace depicting the spontaneous firing rate of a STT neuron before and following bilateral microinjections of vehicle control solution into the MPTA………………........63  Figure 3.13  Example of interspike interval histogram (ISIH) distributions of spike activity of a lumbar STT neuron recorded before and after bilateral microinjections of vehicle control solution into the MPTA of isoflurane‐anesthetized rat preparation...….…..…...….…………………….65  Figure 3.14  Effect of bilateral microinjections of vehicle control solution into the MPTA on the antidromic firing index of STT neurons in the isoflurane‐anesthetized rat preparation...…………….………...……………….68  xi  Figure 3.15  Antidromic firing index of each STT neuron before and after bilateral microinjections of vehicle control solution into the MPTA of isoflurane‐anesthetized rat preparation……………………………...69  Figure 3.16  Effect of bilateral microinjections of vehicle control solution into the MPTA of the isoflurane‐anesthetized rat preparation on sural nerve‐evoked responses of STT neurons………………………...……...………71  Figure 3.17  Example of presynaptic afferent volley recorded in the spinal cord evoked by stimulation of sural nerve......……………...………………………...72  Figure 3.18  Effects of bilateral microinjections of vehicle control solution into the MPTA of the isoflurane‐anesthetized rat preparation on sural nerve‐evoked afferent volley recorded in the lumbar spinal cord of isoflurane‐anesthetized rat preparation………..………………………………..73  Figure 3.19  Effect of bilateral microinjections of vehicle control solution into the MPTA of the isoflurane‐anesthetized rat preparation on sciatic nerve‐ evoked responses of STT neurons.……………………………………………….76  Figure 3.20  Example of presynaptic afferent volley recorded in the spinal cord evoked by stimulation of sciatic nerve..………………...……………………….77  Figure 3.21  Effects of bilateral microinjections of vehicle control solution into the MPTA of isoflurane‐anesthetized rat preparation on sciatic nerve‐ evoked afferent volley recorded in the lumbar spinal cord of the isoflurane‐anesthetized rat preparation………..………………………………..78  xii  Figure 3.22  Effect of bilateral microinjections of pentobarbital into the MPTA of the isoflurane‐anesthetized rat preparation on the spontaneous firing rate of STT neurons………………………...………………...…………….84  Figure 3.23  Spontaneous firing rate of each STT neuron before and after bilateral microinjections of pentobarbital into the MPTA of isoflurane‐ anesthetized rat preparation…………………..…………………….…………...85  Figure 3.24  Example of a continuous ratemeter histogram trace depicting the spontaneous firing rate of a STT neuron before and following the bilateral microinjections of pentobarbital into the MPTA of the isoflurane‐anesthetized rat……….…..…………….……………………………..86  Figure 3.25  Example of interspike interval histogram (ISIH) distributions of spike activity of a lumbar STT neuron recorded before and after bilateral microinjections of pentobarbital into the MPTA of isoflurane‐ anesthetized rat preparation……………...…………….……………...................88  Figure 3.26  Effect of bilateral microinjections of pentobarbital into the MPTA of the isoflurane‐anesthetized rat preparation on the antidromic firing index of STT neurons……………………...……………………………….92  Figure 3.27  Antidromic firing index of each STT neuron before and after bilateral microinjections of pentobarbital into the MPTA of isoflurane‐anesthetized rat preparation…………………………………………93  xiii  Figure 3.28  Effect of bilateral microinjections of pentobarbital into the MPTA of isoflurane‐anesthetized rat preparation on sural nerve‐evoked responses of STT neurons……………………………….……………….………..96  Figure 3.29  Effects of bilateral microinjections of pentobarbital into the MPTA on sural nerve‐evoked afferent volley recorded in the lumbar spinal cord of isoflurane‐anesthetized rat preparation………..…………..…………..97  Figure 3.30  Effect of bilateral microinjections of pentobarbital into the MPTA of isoflurane‐anesthetized rat preparation on sciatic nerve‐evoked responses of STT neurons………………..………….………………………...…101  Figure 3.31  Effect of bilateral microinjections of pentobarbital into the MPTA of isoflurane‐anesthetized rat preparation on sciatic nerve‐evoked afferent volley recorded in the lumbar spinal cord of the isoflurane‐ anesthetized rat preparation…...………………..………………………………102  Figure 3.32  Representative examples of anatomical location of MPTA in the rat brainstem………………………………………………………………………….107  Figure 3.33  Summary of anatomical locations of dye microinjections within the MPTA of rat brain stem…………………...…………….……………………….109  Figure 4.1  Schematic of the hypothetical neural circuitry involved in suppression of STT neuron by pentobarbital microinjections into the rat MPTA…...…...145  xiv  LIST OF SYMBOLS AND ABBREVIATIONS  ANOVA  analysis of variance  AMPA  alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid  AP  anterior‐posterior distance from the bregma  °C  degree Celsius  CD  coefficient of dispersion  cm  centimeter(s)  CNS  central nervous system  CO2  carbon dioxide  CSF  cerebrospinal fluid  CV  coefficient of variation  DRt  dorsal reticular nucleus  DV  ventral distance from the dorsal surface of brain  EEG  electroencephalogram  FI  antidromic firing index  g  gram(s)  GABA  gamma aminobutyric acid  h  hour(s)  Hz  hertz  xv  ID  inner diameter  i.m.  intramuscular  i.v.  intravenous  ISIH  interspike interval histogram  kg  kilogram(s)  kHz  kilohertz  L  liter(s)  L1  first lumbar vertebra/spinal segment  L2  second lumbar vertebra/spinal segment  LC  locus ceruleus  LTD  long‐term depression  LTP  long‐term potentiation  m  meter(s)  M  mole  ML  lateral distance from the midline  MΩ  megaohm(s)  μA  microampere(s)  μg  microgram(s)  μL  microliter(s)  μm  micrometer(s)  xvi  mA  milliampere(s)  mL  milliliter(s)  mm  millimeter(s)  ms  millisecond(s)  min  minute(s)  MPA  medial preoptic area  MPTA  mesopontine tegmental anesthesia area  NaCl  sodium chloride  NE  norepinephrine  nM  nanomol  NMDA  N‐methyl‐D‐aspartic acid  NRGC  nucleus reticularis gigantocellularis  NRM  nucleus raphe magnus  NREM  non‐rapid eye movement  OD  outer diameter  PAD  primary afferent depolarization  PAG  periaqueductal grey  PAH  primary afferent hyperpolarization  PB  pentobarbital  PBS  phosphate buffered saline  xvii  PE  polyethylene  PTg  pedunculopontine tegmental nucleus  PSTH  poststimulus time histogram  REM  rapid eye movement  RVM  rostral ventromedial medulla  s  second(s)  5‐HT  serotonin (5‐hydroxytryptamine)  Sc‐ER  sciatic nerve stimulation‐evoked response(s)  SEM  standard error of mean  SFR  spontaneous firing rate  STT  spinothalamic tract  SS  stainless steel  Su‐ER  sural nerve stimulation‐evoked response(s)  T  threshold intensity  T12  twelfth thoracic vertebra/spinal segment  T13  thirteenth thoracic vertebra/spinal segment  TC  thalamocortical  TMN  tuberomammillary nucleus  Vh  vehicle control solution  VPL  ventral posterior lateral nucleus of thalamus  xviii  ACKNOWLEDGEMENTS As I come to this milestone in the pursuit of knowledge, I cannot help but reflect back and acknowledge all those who have contributed towards completion of this work in one or the other way. First of all, I offer my deepest gratitude to Almighty GOD for showering HIS infinite bounties, graces and mercies on me. Without HIS wishes and blessings, this work could have remained a dream only. I would like to dedicate this work to my parents for their unconditional love, encouragement and to my wife Archana for her immense love and support, who stood with me in every good and bad moment. I would also like to thank my parents‐in‐law for their constant motivation and support. I express my deepest gratitude for my mentor Dr. Peter Soja for his guidance, unstinting  support,  constant  encouragement,  constructive  criticism  and  valuable  suggestions throughout my work. Thank you Peter for introducing me to the exciting world of neuroscience. It has been an enriching experience and privilege working in your laboratory. This work was supported by the grants from U.S. National Institutes of Health (NS041921) and Canadian Institutes of Health Research (CIHR) to Dr. Peter Soja. My sincere thanks to Dr. Shelly A. McErlane, who helped me throughout my study. Thank you Shelly for teaching me aseptic neurosurgical techniques and sharing your vast knowledge of animal care. It has been a delight working with you. I would like to thank my supervisory committee members Drs. Marc Levine (Chair), Sastry Bhagavatula, Anthony Pearson and Nicholas Swindale for providing their critical review of this thesis and the benefit of their experience and expertise.  xix  Special thanks are extended to Dr. Niwat Taepavarapruk for helping me with the schematic of the experimental setup. I am grateful to Dr. Ernest Puil for kindly providing me with pentobarbital used in this thesis. I would also like to convey my deepest gratitude to Dr. Ujendra Kumar for allowing me to use his laboratory facility for the rat brain histology. Special thanks are also extended to Padmesh Rajput for helping me with the histological examinations and imaging of rat brain samples. Special thanks are passed on to Dr. Elissa Strome for sharing some useful tips for microinjection techniques as well as for providing stainless steel cannulae for the drug microinjections. Thanks are also extended to my colleague, Xu (Ervin) Zhu for his help during the initial part of my experiments. I would like to thank the Faculty of Pharmaceutical Sciences at the University of British Columbia for allowing me to use its facilities as well as providing me with Teaching and Research Assistantships. Last but not the least I would like to thank all my well‐wishers, who directly or indirectly contributed to this thesis work.  DHANANJAY NAMJOSHI  xx  DEDICATIONS  Dedicated to  My Parents  &  My Wife and Best Friend  Archana  xxi  General Introduction  CHAPTER 1 GENERAL INTRODUCTION  General anesthesia is a chemically induced, reversible, loss of consciousness that is accompanied by analgesia, atonia, and abolition of autonomic responses. Since the first successful demonstration of ether anesthesia by Morton in 1846 (see, Whalen et al., 2005), general anesthetics have been widely used clinically. Despite over 150 years of clinical practice with general anesthetics, exactly how structurally and pharmacologically distinct substances act to cause general anesthesia is not yet completely understood. Our current knowledge about the mechanism(s) by which general anesthetics act has gradually evolved through in vitro studies that were principally focused on cellular and subcellular targets. The prevailing view is that general anesthetics bind to specific neurotransmitter receptors and inhibit neuronal firing by dampening excitatory synaptic transmission and/or potentiating synaptic inhibition. The state of general anesthesia represents a consortium of different behavioral components including unconsciousness, analgesia, atonia, and attenuation of autonomic responses (Evers et al., 2006). However, exactly which neural circuits are involved in the different components of general anesthesia is still unclear. To correlate the observed clinical effects of general anesthetics to their molecular mechanism(s) of actions, it is necessary to dissect out specific neural structures/networks involved that mediate the different components of general anesthesia, for example analgesia.  1  General Introduction  1.1. Mechanism(s) of Actions of General Anesthetics: From Lipid to Protein Theory  Since substances with a wide array of chemical structures and/or classes can induce general anesthesia, several hypotheses were proposed to link their actions to a single mechanism. Following the first public demonstration of general anesthesia, three theories have emerged to explain the mechanisms of general anesthetic actions. The very first theory of general anesthesia was proposed by the French physiologist, Claude Bernard in 1875. Bernard proposed that general anesthetics act by “reversible coagulation” of chemical constituents of nerves and muscles, in particular, their proteins (Leake, 1971). Bernard’s theory was subsequently refuted for the reasons that coagulation of proteins required a given general anesthetic in a concentration that was much higher than that required for general anesthesia. About thirty years later, Meyer (1899) and Overton (1901) independently proposed the lipid theory of general anesthesia. The Meyer‐Overton lipid hypothesis was based on the observed correlation between the lipid solubility and potency of general anesthetics. Accordingly, the lipid hypothesis proposed that general anesthetics act by perturbation of the lipid membrane. The lipid theory enjoyed a wide, unchallenged acceptance for nearly eight decades.  The lipid theory paradigm was severely challenged by the pioneering work of Franks and Lieb (1984), who, in their ‘protein theory’, proposed that anesthetics could directly bind and modulate membrane proteins. Since then, the research emphasis has shifted from the lipid theory to membrane receptors. Studies over the last two decades have  2  General Introduction revealed several voltage‐gated (Na+, K+ and Ca2+) and ligand‐gated (GABA, glycine, acetylcholine, NMDA and AMPA) ion channels as targets of general anesthetics (for reviews, see Franks and Lieb, 1998; Belelli et al., 1999; Krasowski and Harrison, 1999; Thompson and Wafford, 2001; Campagna et al., 2003; Mashour et al., 2005). The current consensus is that general anesthetics modulate a variety of neurotransmitter channels in the CNS in a distributed manner to cause general anesthesia.  1.2. Central Sites of General Anesthetic Actions  Although much is now known about the molecular and cellular mechanisms of general anesthetics, our understanding of the regions of the CNS affected by them remains elusive. An understanding of the neural networks affected by general anesthetics is required for linking their molecular actions to their observed clinical effects. Along these lines, the effects of various anesthetics have been studied primarily in the brain including various cortical (Alkire et al., 1995; Alkire, 1998; Fiset et al., 1999; Hayton et al., 1999) and sub‐cortical (Guilbaud et al., 1981; Antognini and Carstens, 1999a; Detsch et al., 1999; Ries and Puil, 1999; Alkire et al., 2000) sites. Parallel experiments in rats (Rampil et al., 1993; King and Rampil, 1994; Rampil, 1994; Rampil and King, 1996), cats (Namiki et al., 1980), goats (Antognini and Schwartz, 1993; Antognini and Kien, 1994), and humans (Zhou et al., 1997) indicate that besides the brain, the spinal cord is another important site, for anesthetic action. The principal putative sites in the spinal cord for the actions of general anesthetics are the dorsal horn and motoneuron pools. It is now widely accepted that the unconsciousness and amnesia caused by the general anesthetics are attributed to their supraspinal actions, while  3  General Introduction analgesia and atonia are due to the actions of general anesthetics in the spinal cord (Collins et al., 1995; Antognini, 1997; Antognini and Carstens, 1998, 1999a; Antognini et al., 1999). In addition, many non‐neural sites, including glial cells, cells of the skeleton, cardiomyocytes and cells comprising the immune systems have also been reported as targets of general anesthetics (Urban, 2002).  1.3. Mesopontine Tegmental Anesthesia Area: Potential Central Site for the Induction of General Anesthesia  The studies in the field of general anesthesia so far have assessed global but not specific neural circuits in the CNS where anesthetics might act. Recent intracerebral microinjection studies in rats have provided evidence of discrete loci in the brain that may likely be involved in barbiturate‐induced anesthesia. (Devor and Zalkind, 2001) recently reported on the discovery in conscious rats of a discreet locus in the brainstem pontomesencephalic tegmentum, which might be involved in pentobarbital (PB)‐induced general anesthesia. In this exploratory study, microliter quantities of PB were injected into various brain stem areas of conscious rats while the animals’ anesthesia scores (based on standard behavioral assessment) and electroencephalogram (EEG) were recorded. The authors reported that bilateral microinjections of PB into a focal zone in the mesopontine tegmentum induced a reversible “anesthesia‐like” behavioral state characterized by flaccid muscle atonia, analgesia, loss of consciousness and righting reflexes along with a shift in the EEG waveform pattern from low‐voltage, desynchronized, “fast‐wave” pattern to a high‐voltage, synchronized, “slow‐wave” pattern. In the same study, microinjections of phenobarbital, the  4  General Introduction selective GABAA receptor agonist, muscimol (Johnston, 1996) and alphathesin, a steroid anesthetic, mimicked the effects of PB. Since the cytoarchitectonic boundaries and the target neuron population of this brain stem area were not clearly defined, the authors coined the term mesopontine tegmental anesthesia area (MPTA) to describe this focal site. The authors concluded that the MPTA contains a barbiturate‐sensitive “switch” that may modulate ascending and descending pathways producing the state of anesthesia. The authors have further reported that, the anesthetic actions of PB within the MPTA were highly site‐specific. That is, microinjections of PB in the areas surrounding MPTA either failed to induce anesthesia or induced only mild sedation. Notably, these sites surrounding the MPTA included the periaqueductal grey (PAG), which is involved in morphine‐induced analgesia (Fields et al., 2006) and the pedunculopontine tegmental nucleus (PTg), which plays an important role in regulation of the sleep‐wake cycle (Rye et al., 1987; Steriade and McCarley, 2005).  Surprisingly, in the Devor and Zalkind (2001) study, microinjections of the local anesthetic lidocaine into MPTA failed to reproduce the anesthetic effects of PB. The reason for this discrepancy is not presently known. One possible reason may be that the concentration of the intracerebrally microinjected lidocaine (2%, 0.5 μL volume) used by Devor and Zalkind (2001). In some earlier intracerebral microinjection studies in rats (Aimone and Gebhart, 1986; Ren et al., 1990) a 4% lidocaine (0.5 μL volume) was used to block the activity of local circuit neurons. Thus it may be possible that the lower  5  General Introduction concentration used by Devor and Zalkind (2001) was insufficient to block the MPTA neurons to induce general anesthesia.  Recently, the Devor group has reported that the anesthetic effects of PB microinjections were antagonized by pre‐microinjections of selective GABAA receptor antagonist, bicuculline (Seutin and Johnson, 1999) into the MPTA (Sukhotinsky et al., 2007). This suggests that the anesthesia induced by PB microinjections into the MPTA involves GABAA receptor‐mediated mechanisms. In further neuroanatomical tracing studies, the Devor group has demonstrated that the neurons in the MPTA express GABAA receptors and send axonal projections to various anatomical sites in the higher and lower brain centers as well as spinal cord (Sukhotinsky et al., 2003). The targets of MPTA projections include components of the endogenous motor control systems including the pontine and medullary reticular formation, structures in the rostral ventromedial medulla (RVM), and substantia nigra (Sukhotinsky et al., 2005). MPTA neurons are also reciprocally connected with several supraspinal structures involved in the control of pain transmission, including the periaqueductal grey (PAG), relay nuclei in the RVM, in particular, the nucleus reticularis gigantocellularis (NRGc) (Sukhotinsky et al., 2006). Recently, Sukhotinsky et al. (2007) have reported axonal projections of the MPTA neurons to higher brain centers, which are involved in the control of consciousness. These structures include the intralaminar nuclei of the thalamus, the hypothalamus, the cholinergic PTg, the laterodorsal tegmental nucleus, and forebrain (Sukhotinsky et al., 2007). Interestingly, there are almost no MPTA projections to the thalamic sensory relay nuclei (Sukhotinsky et al., 2007). Finally, MPTA neurons send  6  General Introduction distinct axonal projections to the dorsal and ventral horns of the spinal cord (Sukhotinsky et al., 2006; Reiner et al., 2007; Sukhotinsky et al., 2007). Thus, microinjection of barbiturate anesthetics into the MPTA may inhibit local circuit GABAergic neurons, which in turn, modulate the activities of various target regions of the MPTA in the brain and spinal cord inducing the behavioral state of general anesthesia.  In a recent study, Voss et al. (2005) have corroborated the findings of Devor and Zalkind (2001) by reporting that microinjections of the short‐acting barbiturate anesthetic, thiopental into the MPTA of conscious rats induced general anesthesia‐like state. In the same study, microinjections of non‐barbiturate anesthetic propofol into the MPTA failed to induce general anesthesia. This finding by Voss et al. (2005) is interesting, given that propofol, like barbiturate anesthetics, acts by enhancing the inhibitory GABAergic neurotransmission (Trapani et al., 2000; Irifune et al., 2003). It may be possible, that the dose of propofol microinjected into the MPTA may have been insufficient to induce general anesthesia. Secondly, propofol may be acting by engaging neurons other than the MPTA neurons. Alternately, this may also be attributable to the mechanisms by which propofol and barbiturates modulate the kinetic properties of GABAA receptor‐Clˉ channel. While at anesthetic concentrations, both the barbiturates and propofol increase the Clˉ flux through the GABAA receptor channel, barbiturates increase the mean channel open time of GABAA receptor without altering the channel conductance (Macdonald and Olsen, 1994). On the other hand, therapeutic concentrations of propofol increase the probability of GABAA receptor Clˉ channel being in the open state (Hara et al., 1993; Orser et al., 1994; Trapani et al.,  7  General Introduction 2000). This raises a question whether MPTA‐mediated induction of general anesthesia is dependent on the way in which the kinetic properties of GABAA receptors are modulated.  From the studies of the Devor group, it appears that MPTA has an important role in the induction of general anesthesia. However, is MPTA the sole site for induction of general anesthesia? One potential way to answer this question is by microinjecting bicuculline, into the MPTA of the same animal model as used by Devor and Zalkind (2001), followed by intravenous (i.v.) administration of anesthetic dose of PB. If i.v. administration of PB following bicuculline microinjections into the MPTA fails to induce a general anesthetic state, it may be logical to suggest that the MPTA plays a significant role in engaging the state of barbiturate‐induced general anesthesia. There are, however parallel studies, which indicate that brain nuclei distinct from the MPTA, may also play important role(s) in general anesthetic unconsciousness. For example, Nelson et al.(2002) recently reported dose‐ dependent sedation and a loss of the righting reflex, a hallmark of anesthesia in animals, after microinjection of muscimol, a GABAA agonist into the tuberomammillary nucleus (TMN) of conscious rats. In the same study, microinjections of PB and propofol in the TMN produced only sedation. These responses were antagonized by the putative GABAA receptor antagonist, gabazine. The TMN is a small area located in the hypothalamus which sends major histaminergic input to the cortex and is known to play a critical role in the sleep‐wake cycle (Lin et al., 1990; Wada et al., 1991). Interestingly, the MPTA has very little connectivity with the TMN (Sukhotinsky et al., 2007). Thus these two brain areas, i.e., MPTA and TMN, may be acting independently in the control of consciousness. In a similar type of study,  8  General Introduction microinjections of PB into the medial preoptic area (MPA) of rat brain induced sleep with reduced sleep onset latency and increased non‐rapid eye movement (NREM) sleep as well as total sleep time (Mendelson, 1996). It is not known presently if any neural connectivity exists between MPA and MPTA.  1.4. The Spinothalamic Tract  The spinothalamic tract (STT) is the most important and extensively studied ascending sensory pathway in the anterolateral quadrant of the spinal cord projecting to the thalamus (Hodge and Apkarian, 1990; Willis and Westlund, 1997). STT neurons receive and relay nociceptive, tactile, and thermal information from the periphery to the higher centers in the brain (Willis, 1985; Willis and Westlund, 1997; Willis and Coggeshall, 2004). In addition, the STT has also been implicated in postural changes and locomotion (Menetrey et al., 1984). The distribution of the cell bodies of STT neurons in the spinal cord is species‐specific. Studies in the rat have shown that majority of the STT neurons are located in the nucleus proprious, deep dorsal horn, ventromedial zone and dorsomedial ventral horn (Giesler et al., 1979; Giesler et al., 1981a; Yezierski and Bowker, 1981). The axons of the majority of rat STT neurons decussate initially towards the ventral funiculus and then migrate to the lateral funiculus while ascending to the contralateral thalamus. In rodent brain, the major termination site for STT axons is the ventral posterior lateral (VPL) nucleus of the thalamus (Willis, 1985; Hodge and Apkarian, 1990). STT axons also send collaterals to the medullary reticular formation (Kevetter and Willis, 1982), parabracheal area (Hylden et al., 1989) and periaqueductal gray (Kevetter and Willis, 1983; Liu, 1986; Harmann et al., 1988). STT  9  General Introduction neurons receive a variety of both somatic and visceral afferent inputs. This has been shown by electrically stimulating visceral, cutaneous, and motor nerves (Foreman et al., 1975; Chung et al., 1979; Ammons, 1989).  1.5. Study Rationale  The intracerebral microinjection studies reviewed here suggest that certain neural foci within the brain may play important roles in the generation of unconsciousness due to sleep or general anesthesia. The discoveries of discrete intracerebral foci, when modulated by an anesthetic drug to evoke a behavioral anesthetic state, have challenged the contemporary view that general anesthetics act in a non‐site‐specific manner by ubiquitous inhibition of CNS neurons. The Devor and Zalkind (2001) study, however, was based on the behavioral assessment of the animals. The neurophysiological basis of PB microinjection‐induced anesthesia is not known. The purpose of the study presented in this thesis was to partially address this issue.  The two very important components that are considered as part of the state of general anesthesia are analgesia and atonia. In the microinjection study of Devor and Zalkind (2001), the behavioral assessment of the animal showed that presence of PB in the MPTA caused both analgesia and atonia. However, it may very well be that the analgesia reported in this study was a consequence of the animal’s failure to elicit nociceptive motor responses to the noxious stimuli due to atonia. Accordingly, there is lack of sound evidence that microinjections of PB into the MPTA caused analgesia. One way to address this  10  General Introduction question is by assessing the physiological properties of sensory tract neurons in the spinal cord after PB microinjections into the MPTA.  The afferent fibers carrying sensory (and nociceptive) information from the peripheral tissues make synaptic connectivities with several second‐order ascending pathways in the spinal cord. These ascending pathways, in turn, transmit the sensory signals to the higher brain centers. As mentioned previously, suppression of noxious stimuli‐induced responses by most of the general anesthetics (inhalational, barbiturates, propofol and nitrous oxide) is thought to be due to their direct actions on the spinal cord (Collins et al., 1995; Antognini and Carstens, 1998). Thus, it may be possible that general anesthetic‐induced analgesia is due to the suppression of the spinal ascending pathways (e.g., the STT), which transmit nociceptive input from the periphery to the brain. Surprisingly, there are few, if any, studies assessing the anesthetic‐induced changes in the physiological characteristics of ascending spinal sensory tract neurons.  Soja et al. (2002) have reported that intravenous injection of thiopental reversibly suppressed the spike activities of electrophysiologically identified (proprioceptive) dorsal spinocerebellar tract (DSCT) and (nociceptive) spinoreticular tract (SRT) neurons in the chronic cat preparation. Their study was unique because it examined the activity of DSCT and SRT neurons in the awake, unanesthetized animal, free from recent surgery. The effects of the anesthetic, thiopental were examined on DSCT and SRT neurons under near‐natural conditions. The results obtained by Soja et al. (2002) indicate that barbiturate anesthetics suppress ascending non‐nociceptive and nociceptive transmission through the spinal cord.  11  General Introduction However, since, the anesthetic was delivered through the i.v. route it may have acted in other parts of the CNS besides the spinal sensory neurons, perhaps even the MPTA (Devor and Zalkind, 2001).  The PB microinjection studies reported by Devor and Zalkind (2001) and the electrophysiological studies reported by Soja et al. (2002), thus, present a unique provenance for studying the effects of intracerebrally administered barbiturate anesthetics on the activity of spinal nociceptive neurons. Accordingly, in the present thesis, the excitability of identified spinothalamic tract (STT) neurons was assessed before, during and after microinjections of PB into the MPTA of the rat brain.  1.6. Research Hypothesis  The research hypothesis for the present study was: “Bilateral microinjections of pentobarbital into the mesopontine tegmental anesthesia area (MPTA) of the rat brain, suppresses sensory inflow through the spinothalamic tract.”  1.7. Research Objectives  The overall objective of the study reported in this thesis was to assess the electrophysiological parameters of identified spinothalamic tract (STT) neurons of the rat, namely, the spontaneous discharge, antidromic excitability and afferent nerve stimulation‐ evoked responses after bilateral microinjections of PB into the MPTA. For the purpose of  12  General Introduction this study, the highest effective concentration of 200 μg/μL (800 nM) with the largest volume of 1 μL/side of PB, which induced a behavioral state of general anesthesia in conscious rats (Devor and Zalkind, 2001) was used. In the control studies, PB‐free vehicle control solution (Vh, 1 μL/side) was microinjected into the MPTA. Since the cytoarchitectonic boundaries of MPTA are not yet clearly defined, the stereotaxic coordinates referring to the MPTA zone in this study were derived from the information of the surrounding structures reported by Devor and Zalkind (2001) and the stereotaxic atlas of the rat brain (Paxinos and Watson, 2007), which together, corroborated with those reported in recent papers (Voss et al., 2005; Sukhotinsky et al., 2006). A brief detail of each electrophysiological parameter is given below.  Electrophysiological Parameter 1: Assessment of Spontaneous Firing Rate  The spontaneous spike activity of a STT neuron is defined as the cell’s background spike activity in the absence of any stimulus. Thus, it would be useful to determine if bilateral microinjections of PB into the MPTA modulate the ongoing afferent input and rhythmic properties of individual STT neurons. In this experimental paradigm, once the STT cell was antidromically identified and confirmed, its spontaneous spike activity was recorded before and at specified time intervals after microinjections of PB/Vh into the MPTA. Samples of recorded spike activity were analyzed to determined mean spike rate, interspike interval, coefficient of variation, and coefficient of dispersion. These two latter parameters are useful for detecting indirectly whether changes in spontaneous firing rate due to drug actions are  13  General Introduction also accompanied by changes in spike patterns (Cocatre‐Zilgien and Delcomyn, 1992; Soja et al., 1996).  Electrophysiological Parameter 2: Assessment of Antidromic Firing Index.  The firing index (FI) is the probability of evoking action potentials in the neuron in response to a consecutive number of stimuli applied to it (Lloyd and McIntyre, 1955). The FI method has long being used for assessing changes in the postsynaptic excitability of motor neurons (Hunt, 1955; Lloyd and McIntyre, 1955; Wilson and Burgess, 1962), group Ia and Ib afferents (Wall, 1958; Willis et al., 1976), tooth pulp afferents (Lisney, 1979; Cairns et al., 1996), and some descending fiber systems (Rudomin and Jankowska, 1981). To assess changes in the FI, the identified STT neurons were “backfired” with 100 consecutive stimuli applied to the VPL nucleus at threshold stimulus intensity. The numbers of antidromic spikes obtained from 75 consecutive stimuli (excluding the collision between the antidromic and orthodromic spikes) were calculated before and after microinjections of PB/ Vh into the MPTA. A decrease in the FI after the microinjections compared to the baseline FI would indicate reduced excitability of the STT cell due to putative somatic hyperpolarization (Wall, 1958, 1962; Lipski, 1981).  Electrophysiological Parameter 3: Assessment of Peripheral Nerve Stimulation‐Evoked Responses  STT neurons receive tactile and afferent nociceptive input from the skin and viscera. Therefore, in this study, the responsiveness of the STT neurons to the afferent peripheral  14  General Introduction nerve stimulation was assessed following the microinjections. In the present experiments, the sciatic (Sc) and sural (Su) nerves were stimulated to evoke synaptic responses recorded in STT neurons. The Sc nerve is the largest nerve originating from the lumbosacral spinal segments and innervates the lower extremities. It is a mixed nerve containing both sensory and motor nerve fibers. The Su nerve is a sensory cutaneous nerve innervating the foot. It joins to the peroneal nerve, which is a branch of the sciatic nerve. The mean response magnitude and latency of the STT neurons, evoked by the stimulation of both of these nerves were studied before and after microinjection of PB into the MPTA. In addition, to determine if microinjections of PB/Vh changed the presynaptic input, the peak‐trough amplitude and latency of orthodromic afferent volley recorded within the spinal cord after peripheral nerve stimulation was analyzed around PB/Vh microinjections.  15  Materials and Methods  CHAPTER 2 MATERIALS AND METHODS  All protocols of the studies reported in this thesis were approved by the University of British Columbia Committee on Animal Care and were carried out in accordance with the national (Canadian Council for Animal Care, 1993) and institutional (University of British Columbia Committee on Animal Care) guidelines. All the experiments were carried out in adult male Sprague‐Dawley rats (290‐590 g), obtained from the animal breeding facility at the University of British Columbia. The animals were housed in standardized conditions with 12:12 h light/dark cycles and ambient temperature (21˚C). The animals were fed on standard laboratory rodent chow and given water ad libitum.  2.1. SURGICAL PROCEDURES  All the surgical procedures were performed using aseptic techniques. During surgery a deep surgical plane of anesthesia was maintained and monitored.  2.1.1. Anesthetic Induction  On the day of the experiment, a rat was weighed and placed in an anesthetizing box (# 500108, Harvard Apparatus, Holliston, MA) that was supplied with a continuous stream of isoflurane (4 %) and nitrous oxide (0.6 L/min), in oxygen (3 L/min). The animal usually lost consciousness within 1‐2 min, which was confirmed by observing loss of righting reflex. Once the animal lost its righting reflex, it was taken out of the chamber and placed on its back on the surgery table. The anesthetic mixture thereafter was delivered through a  16  Materials and Methods custom‐made nose cone and the isoflurane level was lowered to 2.5%. The surgical plane of anesthesia was confirmed with the absence of blink and toe pinch reflexes. The core body temperature was maintained at 37 ± 0.5°C using a feedback‐controlled heating blanket. A lubricated rectal probe was gently inserted about 1 cm into the rectum and used to monitor the body temperature throughout the experiment.  Enrofloxacin (5 mg/kg, i.m.) was administered and lubricating ophthalmic ointment (Lacrilube®) was applied to the eyes to prevent corneal drying. The ointment was re‐applied as needed throughout the experiment. Warm, lactated Ringer’s solution was administered as a continuous drip (10 mL/kg/h) for the entire length of the experiment through a 24 or 26 gauge “over the needle” catheter placed in the lateral tail vein. The drip rate was intermittently checked throughout the experiment. The flow of the Lactated Ringer’s solution was adjusted as needed to compensate for bleeding during surgical manipulations. The portion of the tail, where the intravenous (i.v.) catheter was inserted was checked periodically to ensure proper i.v. catheterization. The skin overlying the neck, left thigh, back, and head was shaved for surgical manipulations. The shaved skin was disinfected by serial application of the disinfectants in the following order: chlorhexidine (4%), isopropyl alcohol (70%), and povidone iodine. Ampicillin sodium (33 mg/kg, slow i.v.) and dexamethasone (0.6 mg/kg, slow i.v.) were slowly administered through the i.v. catheter. Heart rate, blood oxygen levels, and end tidal CO2 were continuously monitored with a pulse oximeter/capnograph (SurgiVet™; Harvard Apparatus, Holliston, MA). The heart rate,  17  Materials and Methods oxygen saturation, end‐tidal CO2, and core body temperature were kept within normal physiological limits at all times.  2.1.2. Tracheotomy  When the animal was in a surgical plane of anesthesia, as confirmed by the absence of a pinch reflex, a partial tracheotomy was performed to allow intubation with an endotracheal tube. First, a vertical midline incision was made in the shaved skin of the ventral aspect of the neck. The subcutaneous tissue and sternohyoideus muscles were longitudinally split by blunt dissection and held apart using a self‐retaining retractor to expose the underlying trachea. The trachea was carefully dissected from the underlying connective tissue. A partial transverse incision was made between two tracheal rings rostral to the ring where the suture threads were tied. A custom made plastic cannula (ID: 0.05ʺ OD: 0.09ʺ), with a beveled end was used for tracheal intubation. The beveled end of the cannula was quickly but carefully inserted through the incision into the trachea. The other end of the cannula was connected to a mechanical respirator (Inspira; Harvard Apparatus, Holliston, MA) using a Y connector. The endotracheal tube was secured with sutures placed distal and proximal to the tracheal incision. The rat was then mechanically ventilated by the respirator after tracheal intubation for the entire length of the experiment. Once the artificial ventilation was started, the isoflurane level was adjusted between 2 ‐ 2.25%, depending on the depth of anesthesia required to complete the remaining surgery. The respiration rate was set between 65‐72 breaths/min. After tracheal intubation, the animal was closely observed for chest expansion. Blood oxygen saturation as well as an end tidal CO2 readings between 1.5% and 2% were  18  Materials and Methods maintained at all times throughout the experiment by adjusting the tidal volume and the respiration rate functions of the ventilator. Once the rat was stabilized after tracheal intubation, the neck incision was closed using tissue adhesive (Vetbond™; 3M Animal Care Products, St. Paul, MN). Thereafter, level of anesthesia was checked by observing heart rate, absence of toe and tail pinch reflexes as well as the eye blink responses.  2.1.3. Sciatic and Sural Nerve Surgery  Following tracheal intubation procedures, the animal was placed on its right side to expose the left sciatic and sural nerves. An incision was made in the shaved skin along the length of the femur. The skin around the incision was carefully separated from the underlying connective tissue and fascia. This was followed by blunt dissection of the biceps femoris muscles, caudal to the femur to expose the sciatic nerve. Once the sciatic nerve was visible, the dissection was continued along the gastrocnemius muscle and the sciatic nerve to expose the sural nerve. Both the nerves were carefully freed from the connective tissue in the popliteal fossa. The incision was irrigated with sterile saline, packed with saline‐soaked gauze, and closed with the tissue adhesive until the end of the thoracolumbar laminectomy.  2.1.4. Thoracolumbar Laminectomy  After peripheral nerve surgery, the animal was placed in prone position. A thoracolumbar laminectomy was then performed to expose the spinal cord between the T13 and L1 segments. The T13 segment was identified by first palpating the last floating rib at the T13 vertebra. Once the T13 vertebra was identified and marked, a midline incision in the shaved  19  Materials and Methods skin of the back was made along length from T12 to L2 vertebrae. The adhering fascia was gently separated from the underlying muscles. Paraspinal incisions were made on the either side of the vertebral column through the muscle layer to the bones extending from T12 to L2 vertebrae. The paravertebral muscles and the underlying connective tissue were carefully dissected from the vertebrae. Then the intervertebral space between L1 and L2 vertebrae was identified. A dorsal laminectomy was performed using microrongeurs, starting from the caudal part of the L1 vertebra to the rostral part of the T13 vertebra. Extreme care was taken to avoid damaging the underlying spinal cord.  After completion of laminectomy, the  exposed spinal segments were covered with a piece of sterile calcium alginate dressing (Curasorb™), soaked in sterile saline. The incision was temporarily closed with tissue adhesive.  2.1.5. Craniotomy  Following laminectomy procedures, the rat was placed in a stereotaxic frame (Kopf® 1430; David Kopf Instruments, Tujunga, CA). The head of the rat was secured firmly to the stereotaxic frame with ear bars and a palate bar. A midline incision was made in the scalp from the frontal bone to the atlanto‐occipital junction to expose the calvarium. The calvarium was exposed and partially etched with 35% phosphoric acid to clearly reveal the bregma (intersection of coronal and sagittal sutures) and lambda. The head was firmly secured in a stereotaxic zero position. Two bilateral trephinations were made in the frontal bones to fix screws for recording the cortical EEG. A unilateral trephination (~1 mm diameter) was made in the parietal bone, contralateral to the site of sciatic nerve surgery for  20  Materials and Methods introducing a microelectrode into the ventral posterior lateral (VPL) nucleus of the thalamus. Finally, two trephinations were also made bilaterally along the central suture in the left and right parietal bones for insertion of microinjection probes into the mesopontine tegmentum anesthesia area (MPTA). After completion of surgical manipulations, all the exposed nerve tissues were covered with warm mineral oil to prevent dehydration.  2.1.6. T13‐L1 Vertebral Immobilization Procedures  Following craniotomy procedures, the laminectomy site was carefully re‐opened. The T12 and L2 vertebrae were tightly secured by two spinal clamps, which in turn, were anchored to the stereotaxic frame, so that the exposed spinal segment was fixed between the two clamps. The clamps were adjusted so that the spinal cord was held level to the frame with no visible respiration‐induced movements. A recording well was constructed around the exposed spinal segments using denture repair acrylic. The dura of the exposed spinal cord was gently incised and reflected away from the recording site. The spinal cord was covered with a small pool of warm mineral oil.  The incision site of peripheral nerve dissection was also re‐opened. The skin flaps surrounding the incision were used to form a pool to contain warm, sterile mineral oil bathing the exposed sciatic and sural nerves. Both these nerves were carefully draped on small bipolar hook electrodes to synaptically activate recorded STT neurons.  21  Materials and Methods  2.2. EXTRACELLULAR RECORDING PROCEDURES  2.2.1. Antidromic Identification of Spinothalamic Tract (STT) Neurons  The experimental setup scheme for extracellular recording and intracerebral microinjections procedures is depicted in Figure 2.1. A Parylene‐C® insulated monopolar tungsten microelectrode (Catalog # 573500, Length: 76 mm, Diameter: 250 μm, 2 MΩ AC, A‐M Systems, Inc., Sequim, WA), secured to a three axis micromanipulator (Kopf® 1460‐61, David Kopf Instruments, Tujunga, CA), was directed into the VPL nucleus of thalamus using the following stereotaxic coordinates: AP: ‐3.0 to –3.5 mm from bregma, ML: 3.0 to 3.3 mm from the midline, and DV: 5.5 to 7 mm from the dorsal surface of the brain (Palecek et al., 1992; Paxinos and Watson, 2007). This electrode was used as the antidromic stimulating electrode for “backfiring” STT neurons. A second monopolar tungsten microelectrode, held by a hydraulic micropositioner (Kopf® 650, David Kopf Instruments, Tujunga, CA), was used for recording the spike activity of L1 spinal neurons.  At the beginning of each recording session, both the thalamic stimulation as well as the spinal recording microelectrodes were checked under a microscope to ensure that electrode tip was perfectly formed according to the manufacturer specifications. If the tip was even slightly bent, a fresh electrode was used. Care was taken that both the stimulating and recording electrodes were held vertically straight against two planes of the micropositioner.  22  Materials and Methods For antidromic identification of lumbar STT neurons, a systematic search of the exposed spinal segment was carried out. Initially, the stimulation electrode was positioned in the thalamus at the following stereotaxic coordinates: AP: ‐3.25 mm, ML: 3.15 mm and DV: 6 mm (references: bregma and dorsal surface of the brain). The “zero” position of the spinal recording electrode was adjusted so that the tip of the electrode just touched the surface of the exposed spinal cord close to the midline and contralateral to the thalamic stimulation site.  For antidromic identification of STT neurons, low‐intensity search stimuli (0.2 ms, 0.67 Hz, 200‐300 μA) were continuously delivered to the VPL nucleus through the stimulating electrode while searching the spinal gray matter for the antidromically activated spikes. While applying search stimuli to the VPL, the recording microelectrode was slowly lowered in 5 μm increments into the exposed spinal segment. The dorsal‐ventral advancement of the recording electrode into the spinal cord was controlled using the Kopf® 650 hydraulic micropositioner. Care was taken not to cause spinal cord compression during the electrode descent. The microelectrode was gradually lowered to a maximum depth of 1800 μm below the dorsal surface of the spinal cord as indicated on the digital display of the microdrive device. If no antidromic spike was detected, the electrode was slowly retracted while searching again for an antidromically propagated spike.  The spinal cord segment was systematically explored in grid‐like fashions from a caudal to rostral direction at 0.5 mm inter‐tract distance. This was carried also out in vertical tracts, 0.5 mm lateral from each other starting at the midline. Antidromic spikes were  23  Materials and Methods usually encountered when such tracking procedures were performed in each anesthetized rat preparation. When an antidromically activated spike was encountered, the recording electrode was moved slowly up or down in 2.5 μm increments until a maximum antidromic spike amplitude was obtained (a minimum signal‐to‐noise ratio of 3:1). The depth of the recording electrode corresponding to the maximum antidromic spike amplitude relative to the surface of the spinal cord was recorded as that depth reading indicated on the digital display of the micropositioner.  The following criteria were used for demonstrating antidromic activation of STT neurons from VPL nucleus: 1. spike responses with a constant antidromic latency (< 0.2 mm variability) as measured from the beginning of the stimulus artifact to the initiation of the antidromic action potential, 2. ability of the STT cell to respond faithfully to short trains (2 ‐ 3) of high‐frequency stimuli (300‐500 Hz), and 3. a collision between orthodromic and antidromically generated action potentials during a critical interval (Lipski, 1981; Palecek et al., 1992; Soja et al., 1995). Each neuron meeting all of the above criteria was therefore identified as a STT neuron and selected for further study of the electrophysiological parameters (Lipski, 1981; Palecek et al., 1992; Soja et al., 1995).  2.2.2. Electroencephalogram  The cortical electroencephalogram (EEG) was continuously monitored and recorded throughout the experiment using standard amplifier (Model 79, Grass Instrument Co, Quincy, MA). The EEG was used as an adjuvant to verify the surgical plane of anesthesia.  24  Materials and Methods The filters on the recording amplifier were set between 10 Hz and 3 kHz (half‐amplitude attenuation). A 60 Hz notch filter was used.  2.2.3. Spike Amplification, Recording and Data Acquisition Procedures  The extracellular spike activity of the STT cell was recorded by passing the signal recording from the microelectrode through a high‐impedance probe and a 60 Hz noise eliminator (Hum Bug, Quest Scientific Instruments Inc., North Vancouver, BC). The signal was then amplified (x10000) and filtered (band‐pass: 100 – 10, 000 Hz) using an AC‐coupled differential amplifier (Model 1800, A‐M Systems, Inc., Sequim, WA). The amplified signal was then routed to an oscilloscope (Model D11, Tektronix, Inc., Beaverton, OR) as well as to a spike processor (Model D130, Digitimer Ltd., Hertfordshire, UK), which was set to detect only the STT action potential waveforms. The amplified and filtered STT spike activity as well as the EEG signals were recorded and digitized “on‐line” into separate waveform channels using a Pentium 4 computer equipped with spike acquisition and analysis software (Spike2®, v 5; Cambridge Electronics Design Limited, Cambridge, UK) and hardware (Power 1401 Plus, Cambridge Electronics Design Limited, Cambridge, UK). The sampling rates for the spike wave data and EEG were 20 kHz and 100 Hz, respectively. Data files of each STT neuron recording were stored on a data server for subsequent off‐line analysis using specialized subroutine scripts.  25  Materials and Methods  2.2.4. Baseline Electrophysiological Recording Procedures  Once the STT cell identification was confirmed and spike amplitude maximized, the thalamic stimulating microelectrode was fixed and held in the position by carefully gluing it to the surrounding cranium with denture acrylic. After the acrylic cured, the micromanipulator holding the stimulating electrode was carefully removed. This procedure was necessary to allow sufficient room on the anterior‐posterior rails of the Kopf stereotaxic frame for two additional micromanipulators required for holding the microinjection cannulae. The STT recorded cell activity was allowed to stabilize for about 20‐30 min before recording of the baseline electrophysiological parameters. Here, the isoflurane levels were first reduced to the lowest possible levels (1.5 ‐ 1.75 %) so that the rat was still maintained at a steady plane of surgical anesthesia as confirmed by the EEG and absence of corneal and pinch reflexes. Initially, the depth of recording, stimulation threshold and latency of the STT cell were measured. The threshold intensity (T) of the cell was defined as the minimum stimulus intensity (0.2 ms pulse duration) required for evoking an antidromic action potential. The electrophysiological parameters recorded during the experimental procedure are described below.  2.2.4.1. Spontaneous Spike Activity  The ongoing spontaneous spike activity of STT cell was recorded for a minimum of 15 min. The spontaneous spike activity was analyzed further with computer software (Spike2®, v 5, Cambridge Electronics Design Limited, Cambridge, UK). A subroutine was employed to  26  Materials and Methods sort the spike templates to ensure that the spike activity of the same STT neuron was recorded throughout the experiment. The average spontaneous spike rate for each STT neuron was calculated using 2 min epochs of the spike activity before and at specified time intervals after intracerebral microinjections of PB/Vh. Interspike interval histograms (ISIH) were plotted for the same 2 min epochs used for the spike activity before and after microinjections. To describe the interspike interval data the following statistical parameters were calculated automatically by Spike2® using custom‐made scripts: median interval, coefficient of variation (CV; CV = standard deviation/mean interval), and coefficient of dispersion (CD; CD = variance/median interval). These later parameters were used to study the spike patterns and potential changes in rhythmic firing properties of STT neurons (Cocatre‐Zilgien and Delcomyn, 1992; Soja et al., 1996). A paired Student’s t test was used to compare the group mean values of spontaneous spike activity at each time point after microinjection with the baseline spontaneous activity as well as the other aforementioned parameters (interspike interval, CD, CV etc.) with the respective baseline values. A value of p ≤ 0.05 was considered to be statistically significant.  2.2.4.2. Antidromic Firing Index  To determine the baseline firing index (FI), triple pulse stimuli (train width: 2 ms) at threshold intensity were applied to the VPL. The interstimulus interval was kept at 1.5 s. A total of 100 consecutive antidromic stimuli were applied to the VPL nucleus. This was done to compensate for the absence of antidromic spikes due to their collision with spontaneously occurring orthodromically propagating spikes. The FI was expressed as a ratio of antidromic  27  Materials and Methods spikes/75 stimulus trials corrected for any spontaneous collisions using the following equation (Lloyd and McIntyre, 1955).  Firing Index (FI) = (Number of antidromic spikes / number of stimulus trials) x 100  A paired Student’s t test was used to compare the group mean FI for all STT neurons tested at each time point after microinjection (discussed further) against the baseline FI. A value of p ≤ 0.05 was considered to be statistically significant.  2.2.4.3. Peripheral Nerve Stimulation‐Evoked STT Responses  Stimulation of the sciatic nerve at the intensities used during the control and test paradigms produces a robust twitch in the hindlimb, which can disturb the position of the recording electrode in the spinal cord. Hence, before the recording of sciatic and sural nerve stimulation‐evoked STT responses, the rat was paralyzed by a single dose of pancuronium bromide (0.03 ‐ 0.3 mg/kg, i.v.). The dose of pancuronium was repeated as required throughout the experiment. The sciatic and sural nerves were stimulated; one nerve at a time, with single shock stimuli (0.1 ms, 0.67 Hz) delivered through the hook electrodes placed on them. Initially, the threshold intensity (T) for stimulation for each nerve was determined as the minimum intensity required to produce a single or short train of orthodromic action potentials (Soja et al., 1993; Soja et al., 1995). The peripheral nerves were then stimulated at the intensities of 2T to 3T for 52 consecutive trials. The evoked STT neuron spike responses of 50 trials were analyzed with specialized software.  28  Materials and Methods Post‐stimulus time histograms (PSTH) were generated with Spike2® software to quantitatively assess the sciatic and sural nerve stimulation‐evoked responses of the STT neurons. Here, PSTHs (bin width: 0.5 ms) were constructed from 50 stimulation trials applied to each of the nerves. A baseline response PSTH as well as PSTH at the four post‐ microinjection time points were constructed. A Spike2® subroutine was used to measure the average response magnitude (mean number of spikes/trial) and response latency (ms) and from the user‐defined epochs of the PSTHs. The response magnitude as well as response latency for each nerve at each post‐microinjection time point (discussed further) were compared with the corresponding baseline values with a paired Student’s t test. A value of p ≤ 0.05 was considered to be statistically significant.  To determine if microinjections of Vh/PB changed the presynaptic afferent input, afferent volleys from both the sural as well as sciatic nerves were studied using peak‐to‐ trough amplitude analysis. Peak‐trough amplitudes of afferent volleys before and after microinjections were compared with a paired Student’s t test. A value of p ≤ 0.05 was considered to be statistically significant.  2.3. DRUG AND CONTROL SOLUTIONS  Pentobarbital sodium (Dumex Medical Surgical Products Ltd., Pickering, ON, Canada) was generously provided by Dr. Ernest Puil (Professor Emeritus, Department of Anesthesiology, Pharmacology and Therapeutics, University of British Columbia). Pentobarbital (PB) was carefully weighed and dissolved in a vehicle composed of 10% ethanol and 20% propylene  29  Materials and Methods glycol (Sigma‐Aldrich Inc., St Louis, MO) in water to make a final concentration of 200 μg/μL (800 nM) (Devor and Zalkind, 2001). PB‐free vehicle was used as control. Both PB and vehicle control solution (Vh) were filtered through a 0.2 μm Millipore filter prior to use. In pilot experiments, it was observed that the pH of the final drug solution and drug‐free vehicle remained between 6.9 and 7.2 in corroboration with Devor and Zalkind (2001). Hence, no pH adjustments were made. Once prepared, the drug solution remained stable without visible crystal formation at room temperature for one week. Hence the same drug solution was used in the experiments that were conducted in the same week. However, if any precipitate was observed, the drug solution was discarded and a fresh solution was made.  2.4. INTRACEREBRAL MICROINJECTIONS PROCEDURE  Once all the baseline parameters of STT neurons were determined, microinjections of either PB or Vh were performed. The detailed procedure used for the microinjections is as follows:  Two custom length (~ 5.5 cm) hypodermic stainless steel (SS) tubes (Catalog # 832400, regular wall, 29‐gauge, ID: 0.0065”, OD: 0.013”, A‐M Systems, Inc., Sequim, WA) were used as cannulae for the microinjections of PB and/or Vh into the MPTA (Figure 2.2). One end of each SS cannula was attached and glued to a polyethylene tube (Intramedic™ PE 20; Becton, Dickinson and Company, Sparks, MD) which, in turn, was attached to a syringe containing saline. Each SS cannula was firmly fastened to a micromanipulator (Kopf® 1460‐ 61, David Kopf Instruments, Tujunga, CA). The two micromanipulators were anchored, one on each of the two anterior‐posterior rails of the Kopf® stereotaxic frame.  30  Materials and Methods Before each experiment, each cannula was visually confirmed to be straight when held in a vertical position perpendicular to the micromanipulator. Initially, the PE tubes and SS cannulae were completely filled with saline. A small volume of air was, then drawn through the SS cannulae to form a small bubble in each of the two PE tubes. According to the protocol, a sufficient quantity of either PB or Vh was then gently drawn into each of the PE tubes via the SS cannulae so that the PB/Vh and saline were separated by the air bubble. The syringe plungers were gently pushed forward to ensure complete filling of the SS cannulae. The initial positions of the bubbles in both the tubes were marked. The bubbles were used for dual purposes: to ensure that the PB/Vh was actually being injected into the brain and to measure the volume injected.  The SS cannulae were gently lowered bilaterally into the brain toward the MPTA using the following stereotaxic coordinates: ‐7.2 to ‐8.0 mm posterior to bregma, ± 1.1 to 1.2 mm lateral to the midline and 7.0 to 7.5 mm ventral to the surface of the brain (Devor and Zalkind, 2001; Voss et al., 2005; Paxinos and Watson, 2007). Extreme care was taken to avoid brain compression while lowering the cannulae. On occasions, bleeding was observed at the onset of cannulae lowering. On such occasions, the probes were slowly taken out and the trephination sites were bathed with a small amount of epinephrine (1 mg/mL) to facilitate hemostasis. Once the bleeding subsided, the trephination sites were rinsed with sterile saline and the cannulae were then carefully lowered into the brain. A total volume of 1 μL/side of PB/Vh was slowly injected bilaterally using a calibrated syringe infusion pump (Model 22, Harvard Apparatus, South Natick, MA). The  31  Materials and Methods injection into the MPTA was confirmed by measuring the displacement of the bubble through a pre‐calibrated distance. The microinjection cannulae were left in place for 15‐30 min after completion of the injections to avoid backtracking of the injection fluid into the probe trajectory (Devor and Zalkind, 2001).  2.5. DRUG/VEHICLE CONTROL‐RESPONSE STUDIES  All the STT neurons reported in this thesis were randomly selected for three kinds of treatment protocols. In the first protocol, the electrophysiological parameters of the identified STT neurons were recorded to establish control baseline activity. Then, Vh (1 μL/side) was microinjected bilaterally into the MPTA. The same parameters of the STT neurons were then measured at pre‐determined time intervals (described further). Each of the STT neuron parameter values at each time interval was then compared with their corresponding baseline values.  In the second treatment protocol, the electrophysiological parameters of identified STT neurons were recorded to establish a control baseline first. Then, 1 μL of PB ( 200 μg/μL) was injected into each side of the MPTA. The same parameters of the STT neurons were then measured at pre‐determined time intervals (described further).  In the third protocol, the electrophysiological parameters of STT neurons were assessed first with the microinjections of Vh followed by PB microinjections. A recovery period of at least 1 hr occurred between the microinjections of Vh and PB.  32  Materials and Methods In this study, temporal responses of the STT neurons after PB and/or Vh microinjection were recorded. The time at which bilateral microinjections of PB and/or Vh in the MPTA were completed was considered as time zero. After completion of the microinjections, each electrophysiological parameter was measured and recorded in the following sequence:  a. The spontaneous firing rate (SFR) of the STT neuron was recorded at the time intervals of  2, 15, 30, and 60 min after completion of the microinjections.  b. The antidromic firing index (FI) of the STT neuron was determined at the time intervals  of 3, 16, 31, and 61 minutes after completion of the microinjections.  c. The sural nerve stimulation‐evoked STT responses (Su‐ER) were recorded at the time  intervals of 3.5, 16.5, 31.5, and 61.5 min after completion of the microinjections.  d. The sciatic nerve stimulation‐evoked STT responses (Sc‐ER) were recorded at the time  intervals of 4.5, 17.5, 32.5, and 62.5 min after the microinjections.  The time periods during which the actions of PB would be expected to occur, were derived from the results obtained from the preliminary studies and those from Devor and Zalkind (2001).  The procedural steps in the recording and microinjection study are summarized the Figure 2.3.  33  Materials and Methods  2.6. BRAIN PERFUSION AND HISTOLOGY  To verify that the microinjections were in the MPTA, at the end of all recording procedures 1 μL of a 2% solution of pontamine blue dye in 4M NaCl was bilaterally microinjected in the same location as that of Vh/PB microinjections. The distance between thalamic stimulation electrode and spinal recording electrode was measured for calculation of the conduction velocity (m/s) of the STT neuron. One hour after the microinjections of the dye, the animal was prepared for perfusion. All the electrodes were removed and the animal was removed from the setup while still being mechanically ventilated and anesthetized. The isoflurane level was deepened to 3‐5%. The animal was placed in a dorsal recumbent position in a shallow tray.  A midline incision was made over the length of the sternum and a sternotomy was performed to allow access to the heart. Self‐retaining retractors were used to hold the sternum open. The pericardium was incised to expose the heart. An i.v. administration line was attached to an i.v. fluid bag containing cold 4% paraformaldehyde. A 16 G x 1” needle was attached to the other end of the administration line. The needle was carefully inserted into the left ventricle of the beating heart and directed towards the aorta. The right atrium was incised and the rat was perfused with the paraformaldehyde solution with the flow rate of 1 drop/s. When all cardiac activity ceased, the ventilator was turned off and anesthesia delivery was terminated. The paraformaldehyde flow was maintained until the fluid draining from the right atrium was clear.  34  Materials and Methods The brain was carefully removed and placed in cold 4% paraformaldehyde and post‐ fixed for at least 24 hours before histological processing. For histological analysis, the brain was washed under a slow and continuous stream of water to wash off any residual paraformaldehyde. Serial coronal sections (40‐50 μm) of the brain were then made with a microtome (Vibratome® 1000 Classic) as free floating slices in phosphate‐buffered saline (PBS). The slices were then counterstained with hematoxylin QS nuclear counterstain (Vector Laboratories Inc., Burlingame, CA). The slices were finally washed with water and fixed on microscopy slides for further microscopic analysis. To locate the dye microinjection sites, the entire brain sections were digitally scanned using a ScanScope®CS scanner system (Aperio Technologies, Inc., Vista, CA) at 40X magnification.  35  Materials and Methods  Figure 2.1 Schematic of the experimental setup for extracellular recording of STT neurons and intracerebral microinjections of pentobarbital and/or vehicle control solutions into the MPTA. In this figure, drawings of transverse sections of thalamus, pons and lumbosacral spinal cord are shown. A Parylene‐C® insulated monopolar tungsten electrode (S) directed towards the ventral posterior lateral (VPL) nucleus of thalamus, contralateral to the sciatic (Sc) and sural (Su) nerve stimulation site, was used to antidromically activate a spinothalamic tract (STT) neuron. The excitability of a STT neuron within the spinal dorsal horn was recorded through another Parylene‐C® insulated monopolar tungsten electrode (R). The identified STT neuron was synaptically activated by two bipolar hook electrodes placed on sciatic (Sc) and sural (Su) nerves. The sensory nerve bundles of the sciatic nerve are shown in continuity with the dorsal root ganglion (DRG). The central branch of the DRG, the primary afferent neuron makes oligosynpatic connections with the STT neuron within the L1 spinal cord dorsal horn. The output from the recording electrode is routed to an amplifier (A), a window discriminator (WD) and spike acquisition hardware (Power 1401) and software (Spike2®) and stored on a computer PC). Two microinjection cannulae (MIC) directed bilaterally into the mesopontine tegmental anesthesia area (MPTA) in the pons were used for PB/Vh microinjections. The PB and/or Vh solutions were injected through the MIC by a microinjection pump.  36  Materials and Methods  D  A B  C  cm  Figure 2.2 Photograph of microinjection cannula system used for intracerebral microinjections of pentobarbital/vehicle control solutions. The cannula consisted of a custom‐made 29‐gauge hypodermic regular wall hypodermic stainless steel tube (A) cut to a length of ~ 5.5 cm. The microinjection cannula was passed through and glued to another 27‐ gauge hypodermic stainless steel tube (B) cut to a length of ~ 2.5 cm. The outer tube, which also provided the structural strength to the cannula, was attached to a polyethylene tube (C). The polyethylene tube, in turn was attached to a syringe (not shown) placed in a microinjection pump (not shown). The probe was held to a Kopf® microelectrode holder (D).  37  Materials and Methods  Antidromic identification of STT neuron  Baseline recording of electrophysiological parameters of STT neuron  Microinjections of PB/Vh into MPTA  Recording of electrophysiological parameters at 2, 15, 30 and 60 min following microinjections  Brain histology  Figure 2.3 Flowchart of the steps involved in electrophysiological parameter recording of STT neuron and microinjections procedures. PB – Pentobarbital, Vh – vehicle control solution  38  Results  CHAPTER 3 RESULTS  The results of the present study are described in the three major partitions as follows.  1. General characteristics of the spinothalamic tract (STT) neurons In this partition the general characteristics of STT neurons are described.  2. Studies of electrophysiological parameters of STT neurons  This partition is divided into three sub‐sections: In the first section, the baseline properties of the three electrophysiological parameters; the spontaneous firing rate, the antidromic firing index, and the peripheral (sural and sciatic) nerve‐evoked STT responses, are reported. In the second sub‐section, the results of effects of bilateral microinjections of the control vehicle solution on the aforementioned properties are described. In the third sub‐section, the results of effects of bilateral microinjections of pentobarbital are described.  3. Histological studies In the last partition, histological results of the microinjection sites in the MPTA are reported.  All values reported in this results chapter are expressed as group mean (± SEM) unless otherwise noted.  39  Results  3.1. SPINOTHALAMIC TRACT NEURONS: GENERAL CHARACTERISTICS  A total of 18 spinothalamic tract (STT) neurons in 18 rats (one neuron/rat) were recorded extracellularly from the L1 spinal segment using the standard electrophysiological recording techniques. Each of the 18 extracellularly recorded STT neurons fulfilled three standard antidromic criteria namely, an invariable latency, high frequency following, and collision between the antidromically and orthodromically propagating action potentials (see Materials and Methods section). An example of a STT neuron meeting all three criteria of antidromicity is shown in the Figure 3.1.  Figure 3.1A shows four superimposed traces of single antidromic stimuli with the corresponding antidromic action potentials. As can be seen in this figure, the antidromic action potentials appear with the same latency after each antidromic stimulus without any “jitter”. If a chemical synapse was involved between the stimulating and the recording electrodes, the antidromic latency would not be constant and the antidromic spikes would not be superimposable.  Figure 3.1B shows the second criterion, i.e., high‐frequency following of the same STT neuron. In this case, short trains of high‐frequency antidromic stimuli were applied to the thalamic electrode. Each train was applied at 1.5 s intervals. The total train width was 9 ms and the inter‐pulse interval within each train was 3 ms. Thus, the frequency of antidromic stimuli was 333 Hz. If a chemical synapse was involved, it would act as a low‐ pass filter (Lipski, 1981), and synaptic transmission would fail at such a high frequency. This would mean that no antidromic responses could be recorded. As can be seen in Fig. 3.1B, the  40  Results STT neuron faithfully followed the high‐frequency stimuli applied to the thalamus indicating that the antidromic action potentials were recorded very close to the soma and that no synapse was involved. The STT neurons reported in this thesis were successfully stimulated using frequencies ranging from 333 Hz to 1.5 kHz, indicating a high safety factor (Lipski, 1981).  The third and the most robust criterion for antidromicity was collision between the antidromically and orthodromically propagating action potentials, as depicted in the Figure 3.3C. If an antidromic stimulus was applied at an interval shorter than a period of occurrence of the orthodromic spike, called the critical delay (Lipski, 1981), the antidromic and orthodromic spikes “collided” and cancelled each other. As a result, no antidromic spike was recorded (*, Figure 3.1C, the lowermost trace). This phenomenon can occur only when the stimulating and the recording electrodes are recording one and the same neuron and no chemical synapse is involved. In many cases, a “collision artifact” was observed, which can often be misconstrued as a nearby STT neuron with very low spike amplitude.  All the antidromically identified STT neurons were recorded in vertical electrode tracks within 0.5 mm lateral from the midline. The mean (± SD) recording depth of all 18 STT neurons was 771.8 ± 396.9 μm (range: 97 – 1528 μm) below the surface of the spinal cord. The depths of thalamic stimulating and spinal recording sites of STT neurons are depicted in the Figure 3.2.  41  Results  Figure 3.1 Criteria used for antidromic identification of STT neuron. 1. invariant latency of the antidromic spikes evoked by single pulse stimuli at the threshold intensity applied to the VPL nucleus of contralateral thalamus (A). The figure shows four superimposed sweeps (note the absence of any “jitter” in the antidromic spike); 2. ability to follow high‐frequency stimulation (333 Hz. 3‐pulse trains lasting 9 ms) applied at stimulus trigger (S) shown at the bottom (B) and 3. collision (*) of antidromically propagating spikes from the thalamus with spontaneous, orthodromic action potentials (C). Insets shown in each figure are voltage and time calibration bars.  42  Figure 3.2 Schematic diagram of estimated thalamic stimulation and spinal recording sites of STT neurons recorded in isoflurane‐ anesthetized rat preparation. The estimated antidromic stimulation sites (black filled circles) in the VPL nucleus of thalamus are shown in the coronal section of the rat brain (A) at the level of ‐3.24 mm from Bregma (Paxinos and Watson, 2007). Figure B depicts cross section of L1 spinal segment of rat (Grant and Koerber, 2004) showing laminae 1‐9 corresponding to the Rexed’s (1952) laminae I‐IX. The estimated contralateral recording sites of STT neurons below the surface of spinal cord are shown with black, filled circles.  43  Results  Legend: VPL – ventral posterior lateral nucleus of the thalamus, VPM: ‐ ventral posterior medial nucleus.  Results The group mean (± SD) antidromic latency of all 18 STT neurons, as measured from the stimulus artifact to the onset of the antidromic action potential, measured 4.4 ± 0.6 ms (range: 3 ‐ 6 ms). The group mean (± SD) conductin distance, as estimated by the post‐ mortem measurement of the separation between the thalamic stimulating and spinal recording microelectrodes, measured 81.4 ± 7.6 mm (range: 60 ‐ 90 mm). This corresponds to an axonal conduction velocity of 18.6 ± 2.5 m/s (range: 14 ‐ 23 m/s). The signal‐to‐noise ratio of the antidromic spike of all the STT neurons included in this study was at least 3:1 and often exceeded 4:1. The spike waveform template of the STT neurons, as monitored and recorded within the computer by the Spike2® software, did not change throughout the length of recording. All the STT neurons included in this study could be stimulated by low‐ intensity antidromic stimuli applied to the thalamus. The mean (± SD) threshold intensity used to “backfire” 18 STT neurons measured 341 ± 143 μA.  A statistical analysis was performed to detect if the conduction velocity of all 18 STT neurons was correlated with their spinal recording depths. As shown in the Figure 3.3, no correlation existed between these two parameters (r(16) = 0.07, p = 0.8, Pearson’s correlation).  3.2. STUDIES OF THE ELECTROPHYSIOLOGICAL PARAMETERS  To examine the supraspinal actions of PB on the overall excitability of the STT neurons, three electrophysiological parameters of STT neurons, namely the spontaneous firing rate (SFR, spikes/s), the antidromic firing index (FI) as well as sural (Su) and sciatic (Sc) nerve stimulation‐evoked STT responses (ER) were measured before and after intracerebral  44  Results bilateral microinjections of either Vh (1 μL/side) or PB, (200 μg/side). This section describes, in detail, the results of the studies of the electrophysiological parameters of the STT neurons before and following Vh/PB microinjections. The section is divided into two major parts: in the first part the results of baseline data of the SFR, FI as well as Su‐ and Sc‐ER are described followed by the results of the data collected following the Vh and/or PB microinjections in the second part.  3.2.1. Three Groups of Spinothalamic Tract Neurons According to the Treatment Protocol All the STT neurons examined in this thesis comprised three groups as determined during post‐hoc analysis with six neurons in each group: Group I, Group II, and Group III. Group I STT neurons were treated only with Vh microinjections while group II STT neurons were treated only with PB microinjections. In the case of Group III STT neurons, after baseline recording, microinjections of Vh were carried out and all the electrophysiological parameters were measured at 2, 15, 30 and 60 min following Vh microinjections. Then, following a recovery period of about an hour after the last time point, PB microinjections were carried out and the parameters were measured at time points described above. Taken together, out of 18 STT neurons, 12 STT neurons were treated with microinjections of Vh and 12 with PB microinjections. As discussed further, there were no significant differences in the baseline values of all the electrophysiological parameters of STT neurons among the three groups (p > 0.05, one‐way ANOVA).  Further details on the STT neuron groups and their treatment protocols are summarized in Figure 3.4.  45  Results  Axonal Conduction Velocity (m/s)  24 22 20  r = 0.07 18 16 14 12 0  200  400  600  800  1000 1200 1400 1600 1800  Spinal Recording Depth (μm)  Figure 3.3 Correlation between axonal conduction velocity and spinal recording depth of STT neurons. The abouve figure depicts scatter plot of axonal conduction velocity (m/s) and the respective spinal recording depths (μm) of 18 antidromically identified STT neurons. The coefficient of correlation (r) was 0.07. Note that no significant correlation exists between the two parameters (p = 0.8, Pearson’s correlation).  46  Results  Measurement of SFR, FI, Sc‐Su ER of STT neuron  Group I (n =6)  Group II (n = 6)  Group III (n = 6)  Vh  PB  Vh  Microinjections  Microinjections  Microinjections  Recording of SFR, FI  Recording of SFR, FI  Recording of SFR, FI  and Sc‐Su ER at 2, 15,  and Sc‐Su ER at 2, 15,  and Sc‐Su ER at 2, 15,  30 and 60 min after Vh  30 and 60 min after PB  30 and 60 min after Vh  microinjections  microinjections  microinjections  Recovery period (~ 60 min)  PB microinjections  Recording of SFR, FI and Sc‐Su ER at 2, 15, 30 and 60 min after PB  End of the experiment  Figure 3.4 Schematic explanation of STT neurons classified into three groups based on the treatment(s) they received. Legend: FI – firing index, PB – pentobarbital, SFR – spontaneous firing rate, Sc‐Su ER ‐ sciatic and sural nerve‐evoked responses, Vh – vehicle control solution  47  Results  3.2.2. ELECTROPHYSIOLOGICAL PARAMETERS AT THE BASELINE  After a STT neuron was isolated and identified, all the electrophysiological parameters (SFR, FI, Su‐ER and Sc‐ER) were measured before the microinjections of Vh and/or PB. The values for each of these parameters served as baseline values for comparison with those obtained after microinjections of Vh and/or PB.  3.2.2.1. Spontaneous Firing Rate  All the STT neurons examined in this thesis were found to be spontaneously active. For determining the baseline spontaneous firing rate (SFR), two distinct epochs from the STT spike waveform of 2 min each were selected. The SFR (in spikes/s) obtained from each of the epochs were averaged to obtain the final baseline SFR. The SFR at the time points following the microinjections were compared against this baseline SFR. The group mean (± SEM) baseline SFR of all the 18 STT neurons was 13.1 ± 2.1 spikes/s (range: 4 ‐ 40). The group mean (± SEM) baseline SFR of Group I, Group II and Group III STT neurons were 8.6 ± 2.1 spikes/s (range: 4 – 18), 11.8 ± 2.9 spikes/s (range: 5 ‐ 22), and 19 ± 4.7 spikes/s (range: 11 – 40), respectively.  A statistical comparison between the baseline SFR of the three groups showed no significant differences (F(2, 15) = 2.7, p = 0.12, one‐way ANOVA).  To determine if baseline SFR of all 18 STT neurons depended on the depth at which they were recorded, a Pearson’s correlation analysis was performed between these two parameters. As shown in the Figure 3.5A no statistically significant correlation existed  48  Results between these two parameters (r(16) = 0.33, p = 0.18, Pearson’s correlation). Similarly, the correlation analysis between the baseline SFR and axonal conduction velocity of all the eighteen STT neurons revealed no significant relationship (Fig. 3.5B, r = 0.39, p > 0.05, Pearson’s correlation).  3.2.2.2. Antidromic Firing Index  The group mean ± SEM baseline FI of all the 18 STT neurons was 87.9 ± 3.3 (range: 53‐100). The group mean (± SEM) baseline FI of Group I, Group II and Group III STT neurons were 82.3 ± 6.1 (range: 63 ‐ 99), 88.9 ± 7.4 (range: 53 ‐ 100) and 93.8 ± 2.1 (range: 86 ‐100), respectively.  The baseline FI of the three groups did not differ significantly from each other (F(2, 15) = 1.04, p = 0.38, one‐way ANOVA).  3.2.2.3. Peripheral Nerve‐Evoked STT Responses  Of 18 STT neurons, no orthodromic responses could be elicited in 2 STT neurons (# 7 and 8) with either sural or sciatic nerve stimulation despite a gradual increase in the stimulus intensity. In case of one STT neuron (# 9), only sural nerve‐evoked responses could be recorded while for another STT neuron (# 11), only sciatic nerve‐evoked responses could be recorded. Thus, sural and sciatic nerve‐evoked responses were recorded and measured in 15 of 18 STT neurons.  49  Results  A. Baseline SFR (spikes/s)  50 40 30  r = 0.33  20 10 0 0  200  400  600  800  1000 1200 1400 1600 1800  Spinal Recording Depth (μm)  B. Baseline SFR (spikes/s)  50 40 30  r = 0.39  20 10 0 0  5  10  15  20  25  Axonal conduction velocity (m/s)  Figure 3.5 Correlation of baseline spontaneous firing rate with spinal recording depth and axonal conduction velocity of STT neurons. The graph in A depicts relationship between the spontaneous firing rate (SFR, spikes/s) of STT neurons (n = 18) and their respective recording depth (μm) within the spinal gray matter. The graph in B shows relationship between the SFR of 18 STT neurons and their respective axonal conduction velocity (m/s). The lines of best fit are drawn through the points. Respective correlation coefficients (r) are denoted on the right side of each graph. Note that there was no significant correlation between these parameters (p > 0.05, Pearson’s correlation).  50  Results  3.2.2.3.1. Sural Nerve‐Evoked STT Responses  Of 18 STT neurons recorded in this project, responses of 15 STT neurons to stimulation of sural nerve were observed. For three STT neurons (# 7, 8 and 11), no response could be elicited with sural nerve stimulation despite a gradual increase in the stimulus intensity.  The threshold intensity for sural nerve stimulation was determined as the minimum stimulus intensity, required to produce a single or short train of orthodromic action potentials. The mean (± SEM) threshold intensity for sural nerve stimulation was 1.8 ± 0.4 mA (range: 0.2 ‐ 5). A typical sural nerve stimulation‐evoked STT response is illustrated in the Figure 3.6B.  Baseline sural nerve stimulation‐evoked responses (Su‐ER) of STT neurons were recorded and measured before the microinjections. The baseline group mean (± SEM) magnitude of the Su‐ER of 15 STT neurons measured 7.4 ± 1.2 spikes/trial (range: 2 ‐ 18). The corresponding group mean (± SEM) response‐to‐latency measured 15 ± 2.8 ms (range: 13 ‐ 18). The group means (± SEM) of the baseline response magnitude of Su‐ER of Group I (n = 5), Group II (n = 6) and Group III (n = 4) STT neurons were 6 ± 2 (range: 2 ‐ 14), 8.8 ± 2.3 (range: 3 ‐ 18) and 7.1 ± 1.1 (range: 4 ‐ 10) spikes/trial, respectively. The baseline values for the response magnitude of the three groups did not differ significantly from each other (F(2, 12) = 0.53, p = 0.6, one‐way ANOVA). The response latencies of Group I, Group II and Group III STT neurons were 14.8 ± 5.9 (range: 13 ‐ 17), 16.1 ± 5.2 (range: 15 ‐ 18) and 14 ± 2.9 ms (range: 13 ‐ 15), respectively. The baseline values for the response latency of the three groups did not differ significantly from each other (F(2, 12) = 0.04, p = 0.96, one‐way ANOVA).  51  Results Previous studies have shown that repetitive application of peripheral stimuli can lead to suppression of spinal neurons, called the habituation phenomenon (Griffin and Pearson, 1967; Macdonald and Pearson, 1979). To test if repetitive stimulation of sural nerve in the present study underwent a habituation phenomenon, the magnitude of synaptic responses of five STT neurons to the first, second, third, fourth and fifth batteries of ten consecutive stimuli as well as to a total battery of fifty consecutive stimuli were compared. As shown in the Figure 3.7 there were no significant differences between the magnitudes of Su‐ER STT responses obtained from five individual batteries of ten stimuli (p > 0.05, one‐ way repeated measures ANOVA). Similarly, when response magnitudes obtained from all the five batteries of ten trials were compare with one battery of fifty stimuli no significant differences were found (p > 0.05, one‐way repeated measures ANOVA). This data indicates that repetitive stimulation of sural nerve did not undergo habituation of evoked responses of STT neurons and thus provided stable baseline response.  52  Results  Figure 3.6 Example of sural nerve‐evoked STT responses. The upper left trace (A) shows antidromic spike of a STT neuron recorded in the L1 spinal segment after single pulse (S) stimulation in the thalamus. The upper right trace (B) shows orthodromic responses of the same STT neuron evoked after single pulse (S) stimulation (0.1 ms, 0.9 mA) of the sural nerve (B). Post stimulus time histograms (bin width: 0.5 ms) of the antidromic spike (50 trials) and sural nerve‐evoked response (1 trial) are shown in the bottom traces (C and D), respectively. The insets in A and B are voltage and time calibration lines.  53  Results 14  Number of spikes/trial  12 10 8 6 4 2 0 1-10  11-20  21-30  31-40  41-50  1-50  Stimulus Trials  Figure 3.7 Absence of habituation of sural nerve‐evoked STT responses. The sural nerve was stimulated using the intensity 2 times the threshold intensity. The first five bars represent the group mean (± SEM) response magnitude (number of spikes/trial) of five STT neurons obtained from five consecutive batteries of sural nerve stimulation, each consisting of 10 stimulus trials. The last bar represents group mean (± SEM) response magnitude of five STT neurons obtained from one battery of sural nerve stimulation consisting of 50 stimulus trials. Note that there were no differences among the magnitude of responses obtained from small number (10) of trials as well as large number (50) of trials (p > 0.05, one‐way repeated measures ANOVA).  54  Results  3.2.2.3.2. Sciatic Nerve‐Evoked STT Responses  The threshold intensity for sciatic nerve stimulation was determined as the minimum stimulus intensity, required to produce a single or short train of orthodromic action potentials. Out of 18 STT neurons, responses of 15 STT neurons were recorded after stimulation of sciatic nerve. In three STT neurons (# 7, 8 and 9), no response could be elicited with sciatic nerve stimulation despite a gradual increase in the stimulus intensity. The mean (± SEM) threshold intensity for sciatic nerve stimulation was 1.4 ± 0.7 mA (range: 0.08 ‐ 10).  A typical Sc nerve stimulation‐evoked response (Sc‐ER) of a STT neuron is illustrated in the Figure 3.8B. Note that the cellular response for this particular neuron consisted of “early” and “late” polysynaptic components, although this was observed for only three STT neurons examined.  The group mean (± SEM) magnitude and latency of the Sc‐ER (at 2 times the threshold intensity) of 15 STT neurons measured 8.1 ± 1.5 spikes/trail (range: 1 ‐ 17) and 44.8 ± 1.8 ms (range: 31 ‐ 54), respectively. The group means (± SEM) of the baseline response magnitudes of Group I (n = 5), Group II (n = 6) and Group III (n = 4) STT neurons measured 6.3 ± 2.3 (range: 3 ‐ 15), 9.7 ± 2.8 (range: 1 ‐ 17) and 8.1 ± 1.2 spikes/trial (range: 5 ‐ 11), respectively. The baseline values for the response magnitude of the three groups did not differ significantly from each other (F(2, 12) = 0.51, p = 0.61, one‐way ANOVA).The group means (± SEM) of the baseline response latencies of Group I, Group II, and Group III STT neurons measured 47.2 ± 2.4 (range: 44 ‐ 54), 42.3 ± 3.6 (range: 31 ‐ 54) and 44.5 ± 0.5 ms (range: 43 ‐ 46), respectively. The baseline values for the response latency of all the three  55  Results groups did not differ significantly from each other (F(2, 12) = 0.78, p = 0.48, one‐way ANOVA).  To determine if repetitive stimulation of Sc nerve in the present study underwent a habituation phenomenon (Griffin and Pearson, 1967; Macdonald and Pearson, 1979), the response magnitude of five STT neurons to the first, second, third, fourth and fifth batteries of ten consecutive stimuli with a battery comprising of fifty consecutive stimuli were compared. As shown in the Figure 3.9 there were no significant differences between the response magnitudes of obtained from five individual batteries of ten stimuli (p > 0.05, one‐ way repeated measures ANOVA) as well as with one battery of fifty stimuli (p > 0.05, one‐ way repeated measures ANOVA). This data indicates that repetitive stimulation of Sc nerve did not undergo habituation of evoked responses of STT neurons and thus provided stable baseline response.  56  Results  Figure 3.8 Example of sciatic nerve‐evoked STT responses. The upper left trace (A) shows antidromic spike (arrow) of a STT neuron evoked in the L1 spinal segment after single pulse (S) stimulation in the thalamus. The upper right trace (B) shows orthodromic responses (arrows) of the same STT neuron evoked after single pulse (S) stimulation (0.1 ms, 1.5 mA) of the sciatic nerve. Post stimulus time histograms (bin width: 0.5 ms) of the antidromic spike (50 trials) and sciatic nerve‐evoked response (1 trial) are shown in the bottom traces (C and D), respectively. The insets in A and B are voltage and time calibration lines.  57  Results  Number of spikes/trial  8  6  4  2  0 1-10  11-20  21-30  31-40  41-50  1-50  Stimulus trials  Figure 3.9 Absence of habituation of sciatic nerve‐evoked STT responses. The sciatic nerve was stimulated using the intensity 2 times the threshold intensity. The bars represent the group mean (± SEM) response magnitude (number of spikes/trial) of five STT neurons obtained from five consecutive batteries of sciatic nerve stimulation, each consisting of 10 stimuli as well as a single battery of 50 stimulus trials. Note that there were no differences among the magnitude of responses obtained from small number (10) of trials as well as large number (50) of trials (p > 0.05, one‐way repeated measures ANOVA).  58  Results  3.2.3. EFFECTS OF BILATERAL MICROINJECTIONS OF VEHICLE CONTROL SOLUTION/PENTOBARBITAL  INTO  THE  RAT  MPTA  ON  THE  ELECTROPHYSIOLOGICAL PARAMETERS OF STT NEURONS  3.2.3.1. MICROINJECTIONS OF VEHICLE CONTROL SOLUTION  In the present study, aqueous vehicle containing 10% ethanol and 20% propylene glycol as co‐solvents was used to dissolve PB (Devor and Zalkind, 2001). Ethanol itself can exert differential effects in the CNS, including analgesia (James et al., 1978; Woodrow and Eltherington, 1988). Further, ethanol is shown to suppress the excitability of primary sensory neurons (Oakes and Pozos, 1982; Nieminen, 1987; Gruss et al., 2001). On the other hand, subcutaneous microinjection of ethanol has been shown to increase the sensitivity of dorsal horn sensory neurons (Carstens, 1997). Accordingly, to examine if the vehicle control solution (Vh) itself had any effect on the ongoing as well as evoked spike activity of the STT neurons, all of the three electrophysiological parameters of the STT neurons were compared before and after microinjections of PB‐free vehicle into the MPTA.  3.2.3.1.1. Spontaneous Firing Rate: Vehicle Control Microinjections  To determine if changes in the mean SFR of the STT neurons occurred after microinjections of Vh (1 μL/side) into the MPTA, the SFR at time point (2, 15, 30 and 60 min) following Vh microinjections was compared with the baseline SFR. Figure 3.10 summarizes the group mean (± SEM) SFR of Group I (A, n = 6) and Group III (B, n = 6) STT neurons as well as their  59  Results combined SFR (Vh, n = 12) before and following microinjections of Vh. Comparisons of SFR at each of the time points after Vh microinjections with the respective baseline SFR showed no significant change for STT neurons in the Group I and Group III or when their results were combined (p > 0.05, paired Student’s t test).  The SFR of each STT neuron in the Group I, Group III before and after Vh microinjection are illustrated in the Figure 3.11. Figure 3.12 illustrates a typical rate‐meter histogram trace showing the firing rate of a STT neuron before and following control microinjections.  60  Results  Group III (n = 6)  B.  Group I (n = 6) 16  30  14  25  12  SFR (spikes/s)  SFR (spikes/s)  A.  10 8 6 4 2  20 15 10 5  Vh  Vh  0  0 -30  0  2 15 30 Time Points (min)  SFR (spikes/s)  C.  60  -30  0  2 15 30 Time Points (min)  60  Combined SFR (Groups I and III, n = 12) 25 20 15 10 5  Vh  0 -30  0  2 15 30 Time Points (min)  60  Figure 3.10 Effect of bilateral microinjections of vehicle control solution into the MPTA of isoflurane‐anesthetized rat preparation on the spontaneous firing rate of STT neurons. Time relative to zero is indicated in minutes. Each bar in each graph represents the group mean (± SEM) spontaneous firing rate (SFR, spikes/s). The time point ‐30 min in each graph represents the time point where the baseline SFR was measured before vehicle control solution (Vh; 1 μL/side) microinjections. The graphs in A (open bars) and B (grey bars) show SFR of Group I and Group III STT neurons, respectively. The graph in C (black bars) shows the group mean (± SEM) combined SFR of the Groups I and III STT neurons. Note that the SFR following Vh microinjections in any of the groups as well as in the combined results did not change significantly compared to the baseline SFR (p > 0.05, paired Student’s t test).  61  Results  A. Group I (n = 6)  STT 6 STT 9 STT 10 STT 11 STT 15 STT 18  SFR (spikes/s)  40 30 20 10 0  Vh -30  2 15 30 Time Points (min)  60  B. Group III (n = 6)  STT 7 STT 8 STT 12 STT 14 STT 17 STT 19  50  SFR (spikes/s)  40 30 20 10 0  Vh -30  2 15 30 Time Points (min)  60  Figure 3.11 Spontaneous firing rate of each STT neuron before and after bilateral microinjections of vehicle control solution into the MPTA of isoflurane‐anesthetized rat preparation. The Figure shows spontaneous firing rate (SFR, spikes/s) of each of the STT neurons in Group I (A) and Group III (B). The time point ‐30 min in each graph represents the time point where the baseline SFR was measured before vehicle control solution (Vh; 1 μL/side) microinjections. The downward arrow indicates time zero corresponding to the time of completion of the microinjections.  62  Figure 3.12 Example of a continuous ratemeter histogram trace depicting the spontaneous firing rate of a STT neuron before and following bilateral microinjections of vehicle control solution into the MPTA. The completion of microinjections of vehicle control solution (Vh, 1 μL/side) is denoted by the upward arrow at the bottom of the trace. A sliding average (white line; bin width: 30 s) is superimposed over the rate histogram trace (bin width: 1 s). Spontaneous firing rate (SFR; spikes/s) at the baseline (SFRB) and at 2 min (SFR2), 15 min (SFR15), 30 min (SFR30), and 60 min (SFR60) following Vh microinjections are noted abouve the trace. Note that following microinjections of Vh, the firing rate does not change significantly from the baseline.  Results  63  Results  3.2.3.1.2. Interspike Interval Data: Vehicle Control Microinjections  To determine if the microinjections of the vehicle control solution caused any change in the firing pattern of the STT neurons, the interspike interval histograms (ISIH), as well as the coefficient of variation (CV) and dispersion (CD) were analyzed. To construct ISIHs at each time point, the same data files were used that were used for determining the SFR. The results of the interspike interval data analysis of the Groups I and III STT neurons as well as their combined results are summarized in the Table 3.1.  The ISIH distributions were leptokurtic and in the 10 of the 12 neurons positively skewed. Since the ISIH distributions were skewed, median interval rather than mean interval was measured and analyzed. The overall analysis of the interspike interval data revealed no significant changes after microinjections of Vh.  Similarly, the CV and CD, which are measures of spike train irregularity (Cocatre‐ Zilgien and Delcomyn, 1992; Soja et al., 1996), were not altered by vehicle control microinjections (p > 0.05, paired Student’s t test). Collectively, these data indicate that, microinjections of vehicle control solution into the MPTA did not change the firing pattern of the recorded STT neurons.  An example of ISIH distribution of the spike activity of a STT neuron before and after microinjections of vehicle control solution into the MPTA is represented in Figure 3.13.  64  Figure 3.13 Example of interspike interval histogram (ISIH) distributions of spike activity of a lumbar STT neuron recorded before and after bilateral microinjections of vehicle control solution into the MPTA of isoflurane‐anesthetized rat preparation. Each ISIH was constructed from 2 min epoch of the spike activity recorded before (Baseline) and at each time point (2, 15, 30 and 60 min) following microinjections of vehicle control solution (Vh; 1 μL/side). The median intervals are indicated by dashed vertical lines. Computer‐generated spike trains (5 s) for the respective time points are shown as insets. The time calibration bars (500 ms) are Results  shown below each spike train. Note that the spike pattern of this cell did not change following microinjections of Vh.  65  Results  Interspike Interval Parameter Median Interval (ms)  CV  CD  STT Neuron Group I (n = 6)  Baseline  Minutes after vehicle control (1 μL/side) microinjections 2 15 30 60  28.3 ± 2.3  30 ± 2.7  30.3 ± 3.9  27 ± 5.6  27.7 ± 2.2  III (n = 6)  33.3 ± 7  31.5 ± 4.7  33 ± 10  34.2 ± 7.1  31.1 ± 4.9  Combined I and III  30.8 ± 8.9  33.3 ± 2.3 31.7 ± 3.8  34.6 ± 5.3  29.4 ± 2.5  I (n = 6)  0.8 ± 0.1  0.8 ± 0.1  0.7 ± 0.1  0.8 ± 0.1  1 ± 0.2  III (n = 6)  0.8 ± 0.1  0.8 ± 0.1  0.8 ± 0.1  0.8 ± 0.1  0.8 ± 0.1  Combined I and III  0.8 ± 0.1  0.8 ± 0.1  0.8 ± 0.1  0.8 ± 0.1  0.8 ± 0.0  I (n = 6)  0.1 ± 0.1  0.1 ± 0.1  0.2 ± 0.1  0.2 ± 0.1  0.2 ± 0.1  III (n = 6)  0.1 ± 0.0  0.1 ± 0.1  0.1 ± 0.3  0.1 ± 0.1  0.1 ± 0.1  Combined I and III  0.1 ± 0.0  0.1 ± 0.0  0.3 ± 0.2  0.1 ± 0.0  0.1 ± 0.2  Table 3.1 Summary of effects of bilateral microinjections of vehicle control solution into the MPTA of isoflurane‐anesthetized rat preparation on interspike interval parameters of STT neurons. The table summarizes results of interspike interval analysis of Groups I and III STT neurons as well as their combined data before (baseline) and at the four time points after microinjections of control vehicle solution (1 μL/side). Each parameter is represented as the group mean (± SEM). Note that none of the interspike interval parameters changed significantly following Vh microinjections (p > 0.05, paired Student’s t test). CV: coefficient of variation; CD: coefficient of dispersion  66  Results  3.2.3.1.3. Antidromic Firing Index: Vehicle Control Microinjections  To assess the effects of bilateral microinjections of Vh into MPTA on the excitability of STT neurons, the FI at various time points (2, 15, 30 and 60 min) after the microinjections was compared with the baseline FI.  As shown in Figure 3.14 there was no significant change in the group mean (± SEM) FI of Group I (Fig. 3.14A; n = 6) and Group III (Fig. 3.14B; n = 6) STT neurons after microinjections of Vh when compared to their respective baseline FI (p > 0.05, paired Student’s t test). When the data from these two groups were combined (Fig. 3.14C; n = 12), the mean FI also remained unchanged following microinjections of Vh when compared to the baseline FI (p > 0.05, paired Student’s t test).  The FI of each STT neuron in the Groups I (A; n = 6), and III (B; n = 6) before and following Vh microinjections are presented in the Figure 3.15.  The overall results indicate that the antidromic firing index (FI) and thus the excitability of STT neurons was not significantly altered following microinjections of vehicle control solution into MPTA (p > 0.05, paired Student’s t test).  67  Results  A.  B.  Group I (n = 6)  Group III (n = 6)  100  120  80  100 80 FI  FI  60 40  60 40  20  20  Vh  0  Vh  0 -30  0  2  15  30  60  -30  0  Time Points (min)  C.  2 15 30 Time Points (min)  60  Combined FI (Groups I and III, n = 12) 100 80  FI  60 40 20  Vh  0 -30  0  2 15 30 Time Points (min)  60  Figure 3.14 Effect of bilateral microinjections of vehicle control solution into the MPTA on the antidromic firing index of STT neurons in the isoflurane‐anesthetized rat preparation. Time relative to zero, is indicated in minutes. Each bar in each graph represents the group mean (± SEM) firing index (FI). The time point ‐30 min in each graph represents the time point where the baseline FI was measured before vehicle control solution (Vh; 1 μL/side) microinjections. The graphs in A (open bars) and B (grey bars) show FI of Group I and Group III STT neurons, respectively. The graph in C (black bars) shows the group mean (± SEM) SFR combined FI in Group I and Group III STT neurons. Note that the FI following Vh microinjections in any of the Groups I and III as well as in the combined results did not change significantly compared to the baseline (p > 0.05, paired Student’s t test).  68  Results  A. Group I (n = 6) STT - 6 STT - 9 STT - 10 STT - 11 STT - 15 STT - 18  120 100  FI  80 60 40  Vh  20 -30  2 15 30 Time Points (min)  60  B. Group III (n = 6)  STT - 7 STT - 8 STT - 12 STT - 14 STT - 17 STT - 19  110 100  FI  90 80 70 60  Vh  50 -30  2  15  30  60  Time Points (min)  Figure 3.15 Antidromic firing index of each STT neuron before and after bilateral microinjections of vehicle control solution into the MPTA of isoflurane‐anesthetized rat preparation. The figure depicts antidromic firing index (FI) of each of the STT neurons in Group I (A) and Group III (B). The time point ‐30 min in each graph represents when baseline antidromic firing index FI was measured before vehicle control solution (Vh; 1 μL/side) microinjections. The downward arrow indicates time zero corresponding to the time of completion of the microinjections.  69  Results  3.2.3.1.4. Sural Nerve‐Evoked STT Responses: Vehicle Control Microinjections  Out of 18 STT neurons recorded, sural nerve stimulation could elicit responses in 15 STT neurons. In three neurons, no responses could be elicited by sural (Su) nerve stimulation. Of the 15 STT neurons, 9 STT neurons were assessed for their responses to the stimulation of Su nerve before and following microinjections of Vh.  As shown in Figure 3.16, the microinjection of Vh did not alter the response magnitude as well as the latency of Su‐ER of STT neurons whether it was Group I (Fig. 3.16A), Group III (Fig. 3.16.B), or combined data set (Fig. 3.16C) (p > 0.05, paired Student’s t test).  To examine if microinjections of Vh altered the presynaptic afferent input to the STT neurons, the peak‐trough amplitude analysis of the afferent volley recorded within the spinal cord after Su nerve stimulation were performed. Figure 3.17 illustrates an example of presynaptic afferent volley recorded in the spinal cord followed by a compound action potential evoked by Su nerve stimulation. As shown in the Figure 3.18, both the peak height as well as latency did not change significantly following microinjections of Vh (p > 0.05, paired Student’s t test).  To summarize, both the magnitude as well as latency of responses of STT neurons to stimulation of the Su nerve were not significantly altered following bilateral microinjections of Vh into the MPTA. The afferent volley arising as a consequence of Su nerve stimulation also did not change following Vh microinjections into the MPTA.  70  Results Response Magnitude  Mean Latency Group I (n = 5) 30  18 16 14 12 10 8 6 4 2 0  Mean Latency (ms)  Spikes / Trial  A.  C  Vh  20 15 10 Vh  5 0  -30  0  2  15  30  60  -30  0  Time Points (min)  B.  15  30  60  30  60  30  60  Group III (n = 4) 25 Mean Latency (ms)  8 6 4 2  Vh C  20 15 10 5  Vh  0  0 -30  0  2  15  30  60  -30  0  Time Points (min)  C.  2  15  Time Points (min)  Combined (Groups I and III, n = 9) 14  25 Mean Latency (ms)  12 Spikes / Trial  2  Time Points (min)  10 Spikes / Trial  25  10 8 6  C  4 Vh  2 0 -30  0  2  15  Time Points (min)  30  60  20 15 10 5  Vh  0 -30  0  2  15  Time Points (min)  Figure 3.16 Effect of bilateral microinjections of vehicle control solution into the MPTA of the isoflurane‐anesthetized rat preparation on sural nerve‐evoked responses of STT neurons. Time relative to zero is indicated in minutes. Each bar in each graph represents the group mean (± SEM) value. The time point ‐30 min in each graph represents the time at which the baseline response was measured before microinjections of vehicle control solution (Vh; 1 μL/side). The response magnitudes (spikes/trial) and response latencies (ms) are represented in the left and right panels, respectively. Graphs in A and B represent results of Groups I and III STT neurons, respectively. The graphs in C show combined values of Group I and Group III. In all cases, p > 0.05, paired Student’s t test.  71  Results  B.  Peak height (μV)  A.  Peak latency (ms)  S  0.1 μ V 1 ms  S  0.2 μV 5 ms  Figure 3.17 Example of presynaptic afferent volley recorded in the spinal cord evoked by stimulation of sural nerve. The sural nerve was stimulated at 2 times the threshold intensity, which was the minimum intensity required to produce a single or short train of orthodromic action potentials. The stimulus (S) applied to the sural nerve is followed by afferent volley (enclosed in the box), which in turn, is followed by a compound action potential (A). The trace shown in the upper right (B) is time and amplitude expanded portion of the stimulus and afferent volley.  72  Results Peak‐Trough Amplitude A.  Latency  Group I (n = 5) 4  0.7  0.5  Latency (ms)  Peak Height (μV)  0.6  0.4 0.3 0.2 0.1  2 1  Vh  Vh  0.0  0 -30  0  2 15 30 Time Points (min)  B.  60  -30  4  0.6  3  0.4  2 15 30 Time Points (min)  60  0.2  2 1  Vh  Vh  0.0  0 -30  0  2 15 30 Time Points (min)  C.  60  -30  0  2 15 30 Time Points (min)  60  Combined (Groups I and III, n = 9) 0.7  3.5  0.6  3.0  0.5  2.5  Latency (ms)  Peak Height (μV)  0  Group III (n = 4) 0.8  Latency (ms)  Peak Height (μV)  3  0.4 0.3 0.2 0.1  2.0 1.5 1.0 0.5  Vh  0.0  Vh  0.0 -30  0  2 15 30 Time Points (min)  60  -30  0  2 15 30 Time Points (min)  60  Figure 3.18 Effects of bilateral microinjections of vehicle control solution into the MPTA of the isoflurane‐anesthetized rat preparation on sural nerve‐evoked afferent volley recorded in the lumbar spinal cord of isoflurane‐anesthetized rat preparation. Time relative to zero is indicated in minutes. Each bar in each graph represents the group mean (± SEM) value. The time point ‐30 min in each graph represents the time at which the baseline response was measured before microinjections of vehicle control solution (Vh; 1 μL/side).The peak‐trough amplitudes (μV) and latencies (ms) of afferent volley are shown in the left and right panels, respectively. The graphs in A and B represent results of Groups I and III STT neurons, respectively. The graphs in C show combined values of Group I and Group III. In all cases, p > 0.05, paired Student’s t test.  73  Results  3.2.3.1.5. Sciatic Nerve‐Evoked STT Responses: Vehicle Control Microinjections  Of the 18 STT neurons recorded, sciatic (Sc) nerve stimulation could elicit responses in 15 STT neurons. In three neurons (# 7, 8 and 9), no responses could be elicited by Sc nerve stimulation. Of the 15 STT neurons, 9 STT neurons were assessed for their responses to the stimulation of Sc nerve before and following microinjections of Vh.  As shown in Figure 3.19, microinjections of Vh did not alter the response magnitude or latency of Sc‐ER of STT neurons whether the neurons were from Group I (Fig. 3.19A), Group III (Fig. 3.19B), or combined data sets (Fig. 3.19C) (p > 0.05, paired Student’s t test).  To examine if microinjections of Vh altered the presynaptic afferent input to the STT neurons, the peak‐trough amplitude analysis of the afferent volley recorded within the spinal cord after Sc nerve stimulation were performed. Figure 3.20 illustrates an example of presynaptic afferent volley recorded in the spinal cord followed by a compound action potential evoked by Sc nerve stimulation. The peak‐trough amplitude and latency of the afferent volleys were compared before and after microinjection. As shown in the Figure 3.21, both the amplitude as well as latency of the afferent volley did not change significantly following microinjections of Vh (Fig. 3.21; p > 0.05, paired Student’s t test).  To summarize, both the magnitude as well as latency of responses of STT neurons to stimulation of the Sc nerve were not significantly altered following bilateral microinjections of Vh into the MPTA. The afferent volley arising as a consequence of Sc nerve stimulation also did not change following Vh microinjections into the MPTA.  74  Results Taken together, the results of the present study indicate that bilateral microinjections of vehicle control solution into the MPTA did not alter all the four electrophysiological parameters (i.e. SFR, FI, Su‐ and Sc‐ER) of STT neurons recorded, thus establishing a good control.  Tables 3.2 through 3.4 summarize the results of effects of microinjections of Vh on the electrophysiological parameters of STT neurons recorded in this study.  75  Results  Response Magnitude A.  Response Latency  10  60  8  50  Latency (ms)  Spikes / Trial  Group I (n = 5)  6 4 2  Vh  0  40 30 20 Vh  10 0  -30  0  2 15 30 Time Points (min)  B.  60  -30  0  2 15 30 Time Points (min)  60  2 15 30 Time Points (min)  60  2 15 30 Time Points (min)  60  12  60  10  50  Latency (ms)  Spikes / Trial  Group III (n = 4)  8 6 4 Vh  2 0  40 30 20 Vh  10 0  -30  0  C.  2 15 30 Time Points (min)  60  -30  0  10  60  8  50  Latency (ms)  Spikes / Trial  Combined (Group I and III, n = 9)  6 4 2  Vh  0  40 30 20 Vh  10 0  -30  0  2 15 30 Time Points (min)  60  -30  0  Figure 3.19 Effect of bilateral microinjections of vehicle control solution into the MPTA of the isoflurane‐anesthetized rat preparation on sciatic nerve‐evoked responses of STT neurons. Each bar in each graph represents the group mean (± SEM) value. The time point ‐ 30 min in each graph represents the time at which the baseline response was measured before microinjections of vehicle control solution (Vh; 1 μL/side). The response magnitudes (spikes/trial) and response latencies (ms) are represented in the left right panels, respectively. The graphs in A and B show results of Group I and Group II STT neurons, respectively. The graphs in C show combined values of Group I and Group III. In all cases, p > 0.05, paired Student’s t test.  76  Results  B.  Peak height (μV)  A.  0.1 μV  S Peak latency (ms)  2 ms  S 0.1 μV 10 ms  Figure 3.20 Example of presynaptic afferent volley recorded in the spinal cord evoked by stimulation of sciatic nerve. The sciatic nerve was stimulated at 2 times the threshold intensity, which was the minimum intensity required to produce a single or short train of orthodromic action potentials. The stimulus (S) applied to the sciatic nerve is followed by afferent volley (enclosed in the box), which in turn, is followed by a compound action potential (A). The trace shown in the upper right (B) is time and amplitude expanded portion of the stimulus and afferent volley.  77  Results  Peak‐Trough Amplitude  Latency Group I (n = 5)  1.4  3.5  1.2  3.0  1.0  2.5  Latency (ms)  Peak Height (μV)  A.  0.8 0.6 0.4 Vh  0.2  2.0 1.5 1.0 Vh  0.5 0.0  0.0 -30  0  2 15 30 Time Points (min)  -30  60  0  2  15  30  60  2 15 30 Time Points (min)  60  Time Points (min)  Group III (n = 4) 3.0  3.0  2.5  2.5 Latency (ms)  Peak Height (μV)  B.  2.0 1.5 1.0 0.5  2.0 1.5 1.0 0.5  Vh  Vh  0.0  0.0 -30  0  2 15 30 Time Points (min)  C.  -30  60  0  3.0  3.0  2.5  2.5 Latency (ms)  Peak Height (μV)  Combined (Groups I and III, n = 9)  2.0 1.5 1.0 0.5  2.0 1.5 1.0 0.5  Vh  Vh  0.0  0.0 -30  0  2 15 30 Time Points (min)  60  -30  0  2 15 30 Time Points (min)  60  Figure 3.21 Effects of bilateral microinjections of vehicle control solution into the MPTA of isoflurane‐anesthetized rat preparation on sciatic nerve‐evoked afferent volley recorded in the lumbar spinal cord of the isoflurane‐anesthetized rat preparation. Time relative to zero is indicated in minutes. Each bar in each graph represents the group mean (± SEM) value. The time point ‐30 min in each graph represents the time at which the baseline response was measured before microinjections of vehicle control solution (Vh; 1 μL/side). The peak‐trough amplitudes (μV) and the latencies (ms) of afferent volley are shown in the left and right panels, respectively. The graphs in A and B show results of Group I and Group II STT neurons, respectively. The graphs in C show combined values of Group I and Group III. In all cases, p > 0.05, paired Student’s t test. 78  Results  Group I STT Neurons minutes after vehicle control (1 μL/side)  Electrophysiological Parameter  Baseline  SFR (n = 6)  8.6 ± 2.1  10 ± 4.6  8.5 ± 4.2  8.3 ± 3.43  10.4 ± 3.6  FI (n = 6)  82.3 ± 6.1  74.1 ± 11.3  74.3 ± 11.3  76.7 ± 8.2  84.9 ± 6.9  6±2  7.3 ± 1.8  6.8 ± 1.7  7.1 ± 1.6  11.5 ± 5.4  14.8 ± 5.9  15.7 ± 4.4  15.7 ± 5.8  15 ± 4.6  15.8 ± 10.6  6.3 ± 2.3  6.7 ± 2.2  6.5 ± 2.1  6.5 ± 2.1  4.4 ± 1.2  47.2 ± 2.4  44.6 ± 0.67  43.3 ± 1.1  43.1 ± 1.6  43.2 ± 1  Su-ER Magnitude (n = 5) Su-ER Latency (n = 5) Sc-ER Magnitude (n = 5) Sc-ER Latency (n = 5)  2  microinjections 15 30  60  Table 3.2 Summary of effects of bilateral microinjections of vehicle control solution into the MPTA of isoflurane‐anesthetized rat preparation on the electrophysiological parameters of Group I STT neurons. The results are expressed as the group means (± SEM). The neurons in this group were treated with Vh microinjections (1 μL/side) only. Note that none of the parameters was significantly changed following Vh microinjections (p > 0.05, paired Student’s t test). Legend: SFR: spontaneous firing rate (spikes/s) FI: firing index Su‐ER magnitude: magnitude of sural nerve‐evoked STT response (number of spikes/trial) Su‐ER latency: mean latency of sural nerve‐evoked STT response (ms) Sc‐ER magnitude: magnitude of sciatic nerve‐evoked STT response (number of spikes/trial) Sc‐ER latency: mean latency of sciatic nerve‐evoked STT response (ms)  79  Results  Group III STT Neurons minutes after vehicle control (1 μL/side)  Electrophysiological Parameter  Baseline  SFR (n = 6)  19 ± 4.7  15.4 ± 4.8  14 ± 5.6  18.1 ± 6  18.9 ± 5.8  93.8 ± 2.1  83.8 ± 6.9  86.9 ± 6.7  89.1 ± 5.54  90.4 ± 4.7  7.1 ± 1.1  7.4 ± 2  6.2 ± 2.2  6.8 ± 1.9  7.2 ± 2  14 ± 2.9  14.4 ± 2  14.4 ± 0.4  13.8 ± 5.5  13.9 ± 2.1  8.1 ± 1.2  8.1 ± 1.9  6.7 ± 2.3  5.9 ± 2.1  6 ± 1.8  44.5 ± 0.53  43.4 ± 0.9  43.1 ± 1.9  41.8 ± 1.8  42 ± 1.3  FI (n = 6) Su-ER Magnitude (n = 4) Su-ER Latency (n = 4) Sc-ER Magnitude (n = 4) Sc-ER Latency (n = 4)  2  microinjections 15 30  60  Table 3.3 Summary of effects of bilateral microinjections of vehicle control solution into the MPTA of isoflurane‐anesthetized rat preparation on the electrophysiological parameters of Group III STT neurons. The results are expressed as the group means (± SEM). The STT neurons in this group were treated first with the bilateral microinjections of Vh (1 μL/side) into the MPTA. After recovery, these same neurons were treated with bilateral microinjections of pentobarbital (200 μg/side) into the MPTA. This table shows results obtained following the microinjections of Vh. Note that none of the parameters was significantly changed following Vh microinjections (p > 0.05, paired Student’s t test). Legend: SFR: spontaneous firing rate (spikes/s) FI: firing index Su‐ER magnitude: magnitude of sural nerve‐evoked STT response (number of spikes/trial) Su‐ER latency: mean latency of sural nerve‐evoked STT response (ms) Sc‐ER magnitude: magnitude of sciatic nerve‐evoked STT response (number of spikes/trial) Sc‐ER latency: mean latency of sciatic nerve‐evoked STT response (ms)  80  Results  Combined results of Groups I & III STT Neurons minutes after vehicle control (1 μL/side)  Electrophysiological Parameter  Baseline  SFR (n = 12)  13.8 ± 2.9  12.7 ± 3.3  11.3 ± 3.4  13.2 ± 3.6  15.5 ± 4.1  FI (n = 12)  88 ± 3.5  78.9 ± 6.1  81.1 ± 6.3  82.9 ± 5.1  89.2 ± 1.2  Su-ER Magnitude (n = 9) Su-ER Latency (n = 9) Sc-ER Magnitude (n = 9) Sc-ER Latency (n = 9)  6.5 ± 1.2  7.3 ± 1.2  6.5 ± 1.4  7 ± 1.2  9 ± 2.5  14.5 ± 3.6  15.1 ± 3.4  15.1 ± 3.9  14.5 ± 3.9  14.7 ± 5.7  7.1 ± 1.4  7.3 ± 1.4  6.6 ± 1.5  6.2 ± 1.4  5.2 ± 1.1  43.6 ± 2.3  44.1 ± 3.7  45.6 ± 3.2  47.2 ± 3.9  46.5 ± 3.6  2  microinjections 15 30  60  Table 3.4 Summary of effects of bilateral microinjections of vehicle control solution into the MPTA of isoflurane‐anesthetized rat preparation on electrophysiological parameters obtained by combining the results obtained from Groups I and III STT neurons. The results are expressed as the group means (± SEM). Note that none of the parameters was significantly changed following Vh microinjections (p > 0.05, paired Student’s t test). Legend: SFR: spontaneous firing rate (spikes/s) FI: firing index Su‐ER magnitude: magnitude of sural nerve‐evoked STT response (number of spikes/trial) Su‐ER latency: mean latency of sural nerve‐evoked STT response (ms) Sc‐ER magnitude: magnitude of sciatic nerve‐evoked STT response (number of spikes/trial) Sc‐ER latency: mean latency of sciatic nerve‐evoked STT response (ms)  81  Results  3.2.3.2. MICROINJECTIONS OF PENTOBARBITAL  3.2.3.2.1. Spontaneous Firing Rate: Pentobarbital Microinjections  Figure 3.22 shows the group mean (± SEM) spontaneous firing rate (SFR) of the STT neurons before and at various time points after bilateral microinjections of pentobarbital (PB, 200 μg/side) into the MPTA.  STT neurons in group II (Fig. 3.22A, n = 6) were treated only with PB microinjections. The group mean (± SEM) baseline control SFR of Group II STT neurons before PB microinjections measured 11.8 ± 2.9 spikes/s. For these same neurons, the SFR were significantly reduced to 5.3 ± 1.4 spikes/s at 2 min and to 5.5 ± 1.7 spikes/s at 15 min after microinjections of PB (p < 0.05, paired Student’s t test). This corresponds to a ~ 53% decrease in the spike rate. The reduction in the SFR was reversible within 30 min of PB microinjections. Interestingly, in one STT neuron of this group (# 16), the mean firing rate was abolished 15 min after PB microinjections (Fig. 3.23), which then returned back to the baseline firing rate within 30 min of the microinjections. This effect was not observed for any other of the STT neurons.  The group mean (± SEM) baseline control SFR of Group III STT neurons (Fig. 3.22B, n = 6) before PB microinjections measured 19 ± 4.7 spikes/s. For these same neurons, the SFR was significantly reduced to 12.8 ± 6.1 spikes/s within 2 min after PB microinjections (p < 0.05, paired Student’s t test). This corresponds to a 46% decrease in the spike activity. The reduced SFR was reversed to baseline within 15 min. Interestingly, in the case of Group III  82  Results STT neurons, following recovery of SFR, there was a significant reduction in the mean SFR to 8.7 ± 7.7 spikes/s at the 60 min time point after PB microinjection (p < 0.05, Paired Student’s t test). Note that as reported earlier, in the case of the same Group III STT neurons, the microinjections of Vh did not alter the SFR (see section 3.2.3.1.1).  The combined group mean (± SEM) baseline control SFR of Groups II and III (Fig. 3.22C, n = 12) before PB microinjections measured 15.4 ± 2.8 spikes/s. The combined SFR data showed a significant but reversible decrease in the spike activity to 9.1 ± 3.2 spikes/s at 2 min (p < 0.01, Paired Student’s t test) and 10.7 ± 3.3 spikes/s at 15 min (p < 0.05, Paired Student’s t test) time points after PB microinjections. The reduction in SFR returned to baseline levels within 30 min after PB microinjections.  The SFR of each STT neuron in Group II and Group III is presented in the Figure 3.23. The spike rate of all, but one STT neurons dropped at the 2 min time point and gradually returned towards baseline values. In the case of one neuron (STT neuron # 14), there was a slight (2%) increase in the SFR within 2 minutes of PB microinjections, which was followed by ~ 23% decrease (Fig. 3.23B).  Figure 3.24 illustrates a typical rate‐meter histogram trace depicting the firing rate of a STT neuron before and following PB microinjections. Note that the spontaneous activity almost abolished 15 min following the PB microinjections into the MPTA, although this happened only in the case of this particular STT neuron.  83  Results  A.  B.  Group II (n = 6)  Group III (n = 6) 25  SFR (spikes/s)  SFR (spikes/s)  20 15 10  *  5  *  20  *  15  *  10  PB  5  PB  0  0 -30  0 2 15 30 Time Points (min)  C.  -30  60  0 2 15 30 Time Points (min)  60  Combined (Groups II and III, n = 12)  SFR (spikes/s)  20 15  *  10  *  5 PB 0 -30  0 2 15 30 Time Points (min)  60  Figure 3.22 Effect of bilateral microinjections of pentobarbital into the MPTA of the isoflurane‐anesthetized rat preparation on the spontaneous firing rate of STT neurons. Time relative to zero is indicated in minutes. Each bar in each graph represents the group mean (± SEM) spontaneous firing rate (SFR; spikes/s). The time point ‐30 min in each graph represents the time at which the baseline SFR was measured before microinjections of pentobarbital (PB; 200 μg/side). The graphs in A and B depict the SFR of Group II (open bars) and Group III (grey bars) STT neurons, respectively. The graph in C (black bars) shows the combined SFR of Groups II and III. The asterisks (*) indicate statistically significant reduction in the SFR following PB microinjections compared to the baseline SFR (Paired Student’s t test). The test statistics for significant differences is as follows: Group II (baseline‐ 2 min: t(5) = 3.05, p = 0.03; baseline‐15 min: t(5) = 2.93, p = 0.03); Group III (baseline‐2 min: t(5) = 3.41, p = 0.02; baseline‐60 min: t(3) = 12.65, p = 0.001); combined Groups II and III (baseline‐2 min: t(11) = 3.5, p = 0.005; baseline‐15 min: t(11) = 2.37, p = 0.04).  84  Results  A.  Group II (n = 6) STT - 1 STT - 2 STT - 3 STT - 4 STT - 5 STT - 16  SFR (spikes/s)  30 25 20 15 10 5 0  PB  -30  B.  2 15 30 Time Points (min)  60  Group III (n = 6) STT - 7 STT - 8 STT - 12 STT - 14 STT - 17 STT - 19  SFR (spikes/s)  50 40 30 20 10 0  PB  -30  2 15 30 Time Points (min)  60  Figure 3.23 Spontaneous firing rate of each STT neuron before and after bilateral microinjections of pentobarbital into the MPTA of isoflurane‐anesthetized rat preparation. The figure depicts spontaneous firing rate (SFR; spikes/s) of each STT neuron in Group I (A) and Group III (B). The time point ‐30 min in each graph represents the time at which the baseline SFR was measured before microinjections of pentobarbital (PB; 200 μg/side). The downward arrow indicates time zero corresponding to the time of completion of the microinjections.  85  Figure 3.24 Example of a continuous ratemeter histogram trace depicting the spontaneous firing rate of a STT neuron before and following the bilateral microinjections of pentobarbital into the MPTA of the isoflurane‐anesthetized rat. A sliding average (white line; bin width: 30 s) is superimposed over the rate histogram trace (bin width: 1 s). The time period at which pentobarbital microinjection (PB; 200 μg/side) was completed is denoted by an arrow at the bottom of the trace. Spontaneous firing rate (SFR, spikes/s) at the baseline (SFRB) and at 2 min (SFR2), 15 min (SFR15), and 30 min (SFR30) following PB microinjections is noted on the top of the trace. Note that 15 minutes following PB microinjections the firing rate was nearly abolished and recovered toward the baseline value within 30 minutes. Time calibration bar is shown at the right hand bottom of the trace.  Results  86  Results  3.2.3.2.2. Interspike Interval Data: Pentobarbital Microinjections  To determine if the microinjections of PB caused any change in the firing pattern of the STT neurons, the interspike interval histogram (ISIH), as well as the coefficient of variation (CV) and dispersion (CD) were analyzed. ISIHs were constructed at each time point from the same data files that were used for determining the SFR. The results of the interspike interval data analysis of the STT neurons in the Groups II and III as well as their combined results are shown in the Table 3.5. The interspike interval distributions around PB microinjections were leptokurtic and in 10 of 12 STT neurons positively skewed. The overall analysis of the interspike interval data revealed no significant changes after PB microinjections in any of these statistical parameters (p > 0.05, paired Student’s t test).  Similarly, the CV and CD, which are measures of spike train irregularity, were not significantly altered by PB microinjections (p > 0.05, paired Student’s t test). Collectively, these data indicate that, microinjections of PB into the MPTA did not change the firing pattern of the recorded STT neurons.  An example of ISIH distribution of the spike activity of a STT neuron before and after microinjections of PB is represented in Figure 3.25.  87  Figure 3.25 Example of interspike interval histogram (ISIH) distributions of spike activity of a lumbar STT neuron recorded before and after bilateral microinjections of pentobarbital into the MPTA of isoflurane‐anesthetized rat preparation. Each ISIH was constructed from 2 min epoch of the spike activity recorded before (Baseline) and at each time point (2, 15, 30 and 60 min) following microinjections of pentobarbital (200 μg/side). The median intervals are indicated by dashed vertical lines. Computer‐generated spike trains (5 s) for the respective time points are shown as insets. The time calibration bars (500 ms) are shown below each spike train. The baseline spontaneous firing rate (SFR) of this particular STT neuron was 18.7 spikes/s. The SFR at various time points spikes/s  Results  following PB microinjections was as follows: 2 min = 4.77 spikes/s, 15 min = 5.6 spikes/s, 30 min = 5.1 spikes/s and 60 min = 16.9  88  Results  Interspike Interval Parameter Median Interval (ms)  CV  CD  minutes after pentobarbital (200 μg/side) microinjections 2 15 30 60  STT Neuron Group  Baseline  I (n = 6)  35 ± 6.4  38 ± 3.7  35 ± 5.1  30 ± 13.4  32 ± 19.7  III (n = 6)  40 ± 8.3  40 ± 6.4  41 ± 9.6  43 ± 6.9  38 ± 5.7  Combined I and III I (n = 6)  38 ± 6.8  39 ± 3.7  38 ± 2.9  37 ± 3.4  35 ± 3.4  0.7 ± 0.1  0.8 ± 0.1  0.7 ± 0.1  0.7 ± 0.1  0.8 ± 0.03  III (n = 6)  0.8 ± 0.1  0.8 ± 0.03  0.7 ± 0.1  1.1 ± 0.4  0.6 ± 0.1  Combined I and III I (n = 6)  0.8 ± 0.1  0.8 ± 0.02  0.7 ± 0.1  0.9 ± 0.2  0.7 ± 0.04  0.1 ± 0.02 0.1 ± 0.04  0.1 ± 0.1  0.1 ± 0.02  0.1 ± 0.02  III (n = 6)  0.1 ± 0.1  0.1 ± 0.1  0.1 ± 0.03  0.1 ± 0.1  0.2 ± 0.1  Combined I and III  0.1 ± 0.03 0.1 ± 0.03  0.1 ± 0.03  0.1 ± 0.04  0.1 ± 0.03  Table 3.5 Summary of effect of bilateral microinjections of pentobarbital into the MPTA of isoflurane‐anesthetized rat preparation on interspike interval parameters of STT neurons. The table summarizes results of interspike interval analysis of Groups II and III STT neurons as well as their combined data before (baseline) and at the four time points after microinjections of pentobarbital (200 μg/side). Each parameter is represented as the group mean (± SEM). Note that none of the interspike interval parameters changed significantly following PB microinjections (p > 0.05, paired Student’s t test). CV: coefficient of variation; CD: coefficient of dispersion  89  Results  3.2.3.2.3. Antidromic Firing Index: Pentobarbital Microinjections  To study the effects of bilateral microinjections of PB into the MPTA on the excitability of STT neurons in each treatment group, the mean antidromic firing index (FI) at four time points (2, 15, 30 and 60 min) following microinjections were compared with the baseline FI. These data are presented in Figure 3.26.  The group mean (± SEM) baseline control FI of Group II STT neurons (n = 6) before PB microinjections measured 88.9 ± 7.4. For these same neurons, the group mean (± SEM) FI significantly reduced to 49.2 ± 13 within 2 mins of the PB microinjections (p < 0.05, paired Student’s t test). This corresponds to a 45% reduction in the FI at this time point. The suppression of STT neuron FI after PB microinjections was reversed within 15 min. The results of group II STT neurons are shown in Figure 3.26A.  In the case of Group III STT neurons (n = 6) the group mean (±SEM) baseline control FI before PB microinjections measured 93.8 ± 2.1. Following PB microinjections, the group mean (± SEM) FI of the same STT neurons was significantly reduced to 49.9 ± 20.2 within 15 min (p < 0.05, paired Student’s t test). This corresponds to a 47% decrease in the FI at the same time point. The suppressed FI after PB microinjections recovered to the baseline value within 30 min (Fig. 3.26B). Note that as reported earlier, in the case of the same Group III STT neurons, the microinjections of Vh did not alter the FI (see section 3.2.3.1.2).  90  Results When the FI data from Group II and III STT neurons were combined (n = 12), PB also significantly reduced the FI by 37% to 56.1 ± 11.1 at 2 min time point compared to the baseline FI of 89.2 ± 3.7 (p < 0.05, paired Student’s t test). This reduction was sustained up to the 30 min time point and returned toward the baseline value within 60 min of PB microinjections (Fig. 3.26C).  The FI of each STT neuron in Groups II and III is depicted in the Figure 3.27. The FI was suppressed following PB microinjections in all but 2 STT neurons. In these 2 STT neurons, there was ~ 1 ‐ 8% increase in the FI shortly after PB microinjections.  Taken together, these results indicate that bilateral microinjections of PB into the rat MPTA significantly and reversible suppressed the FI and thus the excitability of STT neurons.  91  Results  B.  A.  Group III (n = 6)  100  100  80  80  *  60  FI  FI  Group II (n = 6)  40 20  *  60 40 20  PB  0  PB  0 -30  0 2 15 30 Time Points (min)  60  -30  0 2 15 30 Time Points (min)  60  C. Combined (Groups II and III, n = 12) 100  FI  80  *  60  *  *  40 20  PB  0 -30  0 2 15 30 Time Points (min)  60  Figure 3.26 Effect of bilateral microinjections of pentobarbital into the MPTA of the isoflurane‐anesthetized rat preparation on the antidromic firing index of STT neurons. Time relative to zero is indicated in minutes. Each bar in each graph represents the group mean (± SEM) antidromic firing index (FI). The time point ‐30 min in each graph represents the time at which the baseline FI was measured before microinjections of pentobarbital (PB; 200 μg/side). The graphs in A and B depict the FI of Group II and Group III STT neurons, respectively. The graph in C shows the FI of Groups II and III combined. The asterisks (*) indicate statistically significant reduction in the FI following PB microinjections compared to the baseline FI. The test statistics (paired Student’s t test) for significant differences is as follows: Group II (baseline‐2 min: t(5) = 3.16, p = 0.05); Group III (baseline‐15 min: t(5) = 2.34, p = 0.04); combined Groups II and III (baseline‐2 min: t(11) = 3, p = 0.01; baseline‐15 min: t(11) = 2.91, p = 0.01; baseline‐30 min: t(11) = 2.39, p = 0.04).  92  Results  A.  FI  Group II (n = 6) STT - 1 STT - 2 STT - 3 STT - 4 STT - 5 STT - 16  120 100 80 60 40 20 0  PB  -30  B.  2 15 30 60 Time Points (min)  Group III (n = 6) 120  STT - 7 STT - 8 STT - 12 STT - 14 STT - 17 STT - 19  100  FI  80 60 40 20 0  PB  -30  2 15 30 60 Time Points (min)  Figure 3.27 Antidromic firing index of each STT neuron before and after bilateral microinjections of pentobarbital into the MPTA of isoflurane‐anesthetized rat preparation. The figure depicts firing index (FI) of each STT neuron in Group I (A) and Group III (B). The time point ‐30 min in each graph represents the time at which baseline FI was measured before pentobarbital (PB; 200 μg/side) microinjections. The downward arrow indicates time zero corresponding to the time of completion of PB microinjections.  93  Results  3.2.3.2.4. Sural Nerve‐Evoked STT Responses: Pentobarbital Microinjections  The sural nerve‐evoked responses (Su‐ER) of 10 out of 18 STT neurons were deemed suitable for testing with PB microinjections. As outlined previously the Su‐ER (2 times the threshold intensity) of STT neurons were recorded and measured at the four time points following PB microinjections. The mean response magnitude and latencies at each of the post‐microinjection time points were compared with the respective baseline readings using paired Student’s t test. The results of the STT neurons in the Groups II and III as well as their combined data are reported below.  The results of effects of PB microinjections on Su‐ER of STT neurons are presented in the Figure 3.28.  The group mean (± SEM) baseline control response magnitude of Group II STT neurons (n = 6) before PB microinjections measured 8.8 ± 2.3 spikes/trial. The group mean (± SEM) response magnitude of the same STT neurons significantly decreased to 4.9 ± 2.1 spikes/trial within 2 minutes following PB microinjections (p < 0.05, paired Student’s t test). This corresponds to a 45% decrease in the response magnitude, which recovered toward baseline values within 15 minutes (Fig. 3.28A, left panel).  The mean latency of Su‐ER of Group II STT neurons was not altered following PB microinjections (Fig. 3.28A, right panel) (p > 0.05, paired Student’s t test).  94  Results In the case of Group III STT neurons (n = 4), there was no significant change in either of the mean magnitude or latency of the Su‐ER following PB microinjections (Fig. 3.28B) (p > 0.05, paired Student’s t test).  When the data of Group II and Group III were combined, the group mean (± SEM) response magnitude was also significantly reduced by 35.5% within 2 min to 6.3 ± 1.7 spikes/trial when compared to the baseline response magnitude of 9.3 ± 1.7 spikes/trial (p = 0.01, paired Student’s t test). The response magnitude remained depressed up to 30 min after which it returned to pre‐microinjection baseline (Fig. 3.28C). There was no post‐PB microinjections significant change in the response latency to sural nerve stimulation in either Group II or III or the combined set of data from Group II and Group III (p > 0.05, paired Student’s t test).  To examine if microinjections of PB altered the presynaptic afferent input to the STT neurons, the afferent volley recorded within the spinal cord after sural nerve stimulation was analyzed. The peak‐trough amplitude and latency of the afferent volleys were compared before and after PB microinjection. As shown in the Figure 3.29, both the peak‐ trough amplitude as well as latency of the sural afferent volley did not change significantly following microinjections of PB (p > 0.05, paired Student’s t test).  These results indicate that bilateral microinjections of PB into the rat MPTA suppressed the magnitude of Su‐ER of STT neurons. The latency of SuER was not altered. Similarly the presynaptic volley arising from sural nerve stimulation was not affected by PB microinjections into the MPTA.  95  Results Response Magnitude  Response Latency Group II (n = 6)  A.  25  14 Mean Latency (ms)  Spikes / Trial  12 10  *  8 6 4 PB  2 0  20 15 10 5  PB  0 -30  0  B.  2 15 30 Time Points (min)  60  -30  0  2 15 30 Time Points (min)  60  2 15 30 Time Points (min)  60  2 15 30 Time Points (min)  60  Group III (n = 4) 14  25 Mean Latency (ms)  Spikes / Trial  12 10 8 6 4 PB  2 0  20 15 10 5  PB  0 -30  0  2 15 30 Time Points (min)  C.  60  -30  0  Combined (Groups II and III, n = 10) 25  Spikes / Trial  10  *  8  Mean Latency (ms)  12  * *  6 4 2  PB  0  20 15 10 5  PB  0 -30  0  2 15 30 Time Points(min)  60  -30  0  Figure 3.28 Effect of bilateral microinjections of pentobarbital into the MPTA of isoflurane‐ anesthetized rat preparation on sural nerve‐evoked responses of STT neurons. Each bar in each graph represents the group mean (± SEM) value. Time relative to zero is indicated in minutes. The time point ‐30 min in each graph represents the time at which baseline responses were measured before microinjections of pentobarbital (PB; 200 μg/side). The response magnitude (spikes/trial) and response latency (ms) are represented in the left and right panels, respectively. The graphs in A and B represent results of Group II and Group III STT neurons, respectively. The graphs in C show the combined data of Group II and Group III STT neurons. The asterisks (*) indicate statistically significant reduction in the response magnitude following PB microinjections compared to the baseline. The test statistics (paired Student’s t test) for significant differences is as follows: Group II (baseline‐2 min: t(5) = 2.72, p = 0.04); combined Groups II and III (baseline‐2 min: t(9) = 3.21, p = 0.01; baseline‐15 min: t(9) = 2.67, p = 0.03; baseline‐30 min: t(9) = 2.6, p = 0.03). 96  Results  Peak‐Trough Amplitude A.  Latency  Group II (n = 6) 5  0.7  4  0.5  Latency (ms)  Peak Height (μV)  0.6  0.4 0.3 0.2  2 1  PB  0.1  3  0.0  PB  0 -30  0  2 15 30 Time Points (min)  60  B.  -30  0  2 15 30 Time Points (min)  60  1.0  1.0  0.8  0.8 Latency (ms)  Peak Height (μV)  Group III (n = 4)  0.6 0.4 0.2 0.0 0  2 15 30 Time Points (min)  C.  0.4 0.2  PB -30  0.6  PB  0.0  60  -30  0  2 15 30 Time Points (min)  60  0.6  0.6  0.5  0.5 Latency (ms)  Peak Height (μV)  Combined (Groups II and III, n = 1))  0.4 0.3 0.2 0.1  0.4 0.3 0.2 0.1  PB  0.0  PB  0.0 -30  0  2 15 30 Time Points (min)  60  -30  0  2 15 30 Time Points (min)  60  Figure 3.29 Effects of bilateral microinjections of pentobarbital into the MPTA on sural nerve‐evoked afferent volley recorded in the lumbar spinal cord of isoflurane‐anesthetized rat preparation. Time relative to zero is indicated in minutes. The time point ‐30 min in each graph represents the time at which baseline responses were measured before PB (200 μg/side) microinjections. Each bar represents the group mean (± SEM) value. Peak‐trough amplitude (μV) and the latency (ms) of afferent volley are shown in the left and right panels, respectively. The graphs in A and B represent results of Groups II and III STT neurons, respectively. The graphs in C show combined results of Group II and III. For all cases, p > 0.5, paired Student’s t test.  97  Results  3.2.3.2.5. Sciatic Nerve‐Evoked STT Responses: Pentobarbital Microinjections  The sciatic nerve‐evoked responses (Sc‐ER) of 10 out of 18 STT neurons were deemed suitable for testing with pentobarbital (PB) microinjections. As outlined previously the Sc‐ ER (2 times the threshold intensity) were recorded and measured at the four time points following PB microinjections. The mean response magnitude and latencies at each of the post‐microinjection time points were compared with the respective baseline readings using paired Student’s t test. The results of the STT neurons in the Groups II and III as well as their combined data are reported below.  The results of Sc‐ER of STT neurons following PB microinjections are graphically summarized in the Figure 3.30.  The group mean (± SEM) baseline control response magnitude of Group II STT neurons (n = 6) before PB microinjections measured 9.7 ± 2.8 spikes/trial. Following PB microinjections, the group mean (± SEM) magnitude of Sc‐ER of Group II (n = 6) STT neurons was significantly reduced by 35% to 6.3 ± 2.7 spikes/trial at 2 min, by 42% to 5.6 ± 2.7 spikes/trial at 15 min and by 41% to 5.7 ± 2.4 spikes/trial at 30 min time points following PB microinjections (p < 0.05, paired Student’s t test). The response magnitude recovered to the pre‐microinjection baseline value with 60 min of PB microinjections (Fig. 3.30A, left panel).  98  Results In the case of Group III STT neurons (n = 4), the mean magnitude as well as latency of the Sc‐ER were not altered following PB microinjections (Fig. 3.30B) (p > 0.05, paired Student’s t test).  When the data of Group II and Group III were combined (n = 10), the group mean (± SEM) baseline response magnitude measured 9.7 ± 1.8 spikes/trial. For this combined data set the response magnitude was also significantly reduced by 26% to 7.2 ± 1.6 spikes/trial at 2 min, by 29% to 6.9 ± 1.7 spikes/trial at 15 min, and by 27% to 7.1 ± 1.7 spikes/trial at 30 min time point following PB microinjections (p < 0.05, paired Student’s t test). The suppressed response magnitude recovered toward the pre‐injection baseline within 60 min of PB microinjection (Fig. 3.30C, left panel). The response latency to sciatic nerve stimulation in either Group II or III or the combined data set of Group II and Group III was not altered by PB microinjections (p > 0.05, paired Student’s t test).  To examine if microinjections of PB altered the presynaptic afferent input to the STT neurons, the afferent volley recorded within the spinal cord after sciatic nerve stimulation was analyzed. The peak‐trough amplitude and latency of the afferent volleys were compared before and after PB microinjection. As shown in the Figure 3.31, both the peak‐ trough amplitude as well as latency of the sciatic afferent volley did not change significantly following microinjections of PB (p > 0.05, paired Student’s t test).  These results indicate that bilateral microinjections of PB into the rat MPTA suppressed the magnitude of Sc‐ER of STT neurons. The mean latency of the evoked  99  Results responses was not altered. Similarly the presynaptic volley arising from sciatic nerve stimulation was not affected by PB microinjections into the MPTA.  The results of effects of microinjections of PB into the MPTA on the four electrophysiological parameters are illustrated in Tables 3.6 through 3.8.  100  Results Response Magnitude A.  Response Latency  Group II (n = 6) 60 Mean Latency (ms)  14 Spikes / Trial  12 10  *  8 6  *  *  4 PB  2 0  0  2 15 30 Time Points (min)  B.  30 20 10  60  PB  -30  0  2 15 30 Time Points (min)  60  Group III (n = 4)  14 Mean Latency (ms)  60  12 Spikes / Trial  40  0 -30  10 8 6 4 PB  2 0  50 40 30 20 10  PB  0 -30  0  2 15 30 Time Points (min)  C.  60  -30  0  2 15 30 Time Points (min)  60  Combined (Groups II and III, n = 10)  14 Mean Latency (ms)  60  12 Spikes / Trial  50  10  *  8  *  *  6 4 PB  2 0  50 40 30 20 10  PB  0 -30  0  2 15 30 Time Points (min)  60  -30  0  2 15 30 Time Points (min)  60  Figure 3.30 Effect of bilateral microinjections of pentobarbital into the MPTA of isoflurane‐ anesthetized rat preparation on sciatic nerve‐evoked responses of STT neurons. Each bar in each graph represents the group mean (± SEM) value. Time relative to zero is indicated in minutes. The time point ‐30 min in each graph represents the time at which baseline responses were measured before microinjections of pentobarbital (PB; 200 μg/side). The response magnitude (spikes/trial) and response latency (ms) are represented in the left and right panels, respectively. The graphs in A and B represent results of Group II and Group III STT neurons, respectively. The graphs in C show combined data of Group II and Group III STT neurons. The asterisks (*) indicate statistically significant reduction in the response magnitude following PB microinjections compared to the baseline magnitude. The test statistics (paired Student’s t test) for significant differences is as follows: Group II (baseline‐2 min: t(5) = 3.66, p = 0.02; baseline‐15 min: t(5) = 4.07, p = 0.01; baseline‐30 min: t(5) = 4.89, p = 0.005); combined Groups II and III (baseline‐2 min: t(9) = 3.32, p = 0.009; baseline‐15 min: t(9) = 3.61, p = 0.006; baseline‐30 min: t(9) = 3.28, p = 0.01).  101  Results  Peak‐Trough Amplitude A.  Latency Group II (n = 6) 5  0.7  4  0.5  Latency (ms)  Peak Height (μV)  0.6  0.4 0.3 0.2  3 2 1  PB  0.1  PB  0  0.0 -30  0  2 15 30 Time Points (min)  -30  60  B.  0  2 15 30 Time Points (min)  60  2 15 30 Time Points (min)  60  1.0  5  0.8  4 Latency (ms)  Peak Height (μV)  Group III (n = 4)  0.6 0.4 0.2  3 2 1  PB  PB  0  0.0 -30  0  2 15 30 Time Points (min)  C.  -30  60  0  Combined (Groups II and III, n = 10) 4  0.6  Latency (ms)  Peak Height (μV)  0.5 0.4 0.3 0.2 0.1  3 2 1  PB  PB  0.0  0 -30  0  2 15 30 Time Points (min)  60  -30  0  2 15 30 Time Points (min)  60  Figure 3.31 Effect of bilateral microinjections of pentobarbital into the MPTA of isoflurane‐ anesthetized rat preparation on sciatic nerve‐evoked afferent volley recorded in the lumbar spinal cord of the isoflurane‐anesthetized rat preparation. Each bar in each graph represents the group mean (± SEM) value. Time relative to zero is indicated in minutes. The time point ‐30 min in each graph represents the time at which baseline responses were measured before microinjections of pentobarbital (PB; 200 μg/side). The peak‐trough amplitude (μV) and the latency (ms) of afferent volley are shown in the left and right panels, respectively. The graphs in A and B depict results of Group II and Group III STT neurons, respectively. The graph in C shows combined results of Group II and Group III. For all cases p > 0.05, paired Student’s t test.  102  Results  Group II STT Neurons Electrophysiological Parameter  Baseline  SFR (n = 6)  11.8 ± 2.9  FI (n = 6)  88.9 ± 7.4  Su-ER Magnitude (n = 6) Su-ER Latency (n = 6) Sc-ER Magnitude (n = 6) Sc-ER Latency (n = 6)  8.8 ± 2.3  minutes after pentobarbital (200 μg/side) microinjections 15 30  2  16.1 ± 5.2 9.7 ± 2.8 42.3 ± 3.6  60  9.7 ± 1.3  12.1 ± 3.5  66.8 ± 14.1  78.5 ± 14.2  78 ± 12.2  *  5.8 ± 2.2  5.9 ± 2.3  10.2 ± 2.3  15.7 ± 3.7  16 ± 4.8  15.9 ± 6.6  16.6 ± 4.6  *  5.6 ± 2.4  *  9.6 ± 2.6  41.2 ± 6.14  44.6 ± 5.6  5.3 ± 1.4  *  5.5 ± 1.9  *  49.2 ± 13 4.9 ± 2.1  6.3 ± 2.7  43.2 ± 5.8  *  *  43.4 ± 4.8  5.7 ± 2.4  Table 3.6 Summary of effects of bilateral microinjections of pentobarbital into the MPTA of the isoflurane‐anesthetized rat preparation on the electrophysiological parameters of Group II STT neurons. The results are expressed as the group means (± SEM). The neurons in this group were treated with pentobarbital microinjections (200 μg/side) only. The asterisks (*) indicate statistically significant reduction in the electrophysiological parameter following pentobarbital microinjections compared to its respective baseline value (p < 0.05, Paired Student’s t test). Legend: SFR: spontaneous firing rate (spikes/s) FI: firing index Su‐ER magnitude: magnitude of sural nerve‐evoked STT response (number of spikes/trial) Su‐ER latency: mean latency of sural nerve‐evoked STT response (ms) Sc‐ER magnitude: magnitude of sciatic nerve‐evoked STT response (number of spikes/trial) Sc‐ER latency: mean latency of sciatic nerve‐evoked STT response (ms)  103  Results  Group III STT Neurons Electrophysiological Parameter  Baseline  SFR (n = 6)  19 ± 4.7  FI (n = 6) Su-ER Magnitude (n = 4) Su-ER Latency (n = 4) Sc-ER Magnitude (n = 4) Sc-ER Latency (n = 4)  minutes after pentobarbital (200 μg/side) microinjections 15 30  2 12.8 ± 6.1  *  15.9 ± 5.8  15.9 ± 4.6  60 8.7 ± 7.7  *  51.3 ± 20.9  65.1 ± 12.4  8.7 ± 2.6  8.6 ± 1.3  8.9 ± 2.4  14.5 ± 0.5  14.4 ± 0.2  14.5 ± 1.6  14.8 ± 1.8  9.7 ± 2.4  8.5 ± 2.6  8.8 ± 2.5  9.2 ± 2.2  9.4 ± 2.5  44.5 ± 0.5  45.5 ± 0.9  42.9 ± 1.2  43.5 ± 0.8  44.8 ± 1.1  93.8 ± 2.1  63 ± 19.4  10 ± 2.8  8.4 ± 2.7  14.3 ± 4.6  49.9 ± 20.2  *  Table 3.7 Summary of effects of bilateral microinjections of pentobarbital into the MPTA of isoflurane‐anesthetized rat preparation on the electrophysiological parameters of Group III STT neurons. The results are expressed as the group means (± SEM). The STT neurons in this group were treated first with the bilateral microinjections of vehicle control solution (1 μL/side) into the MPTA. After recovery, these same neurons were treated with bilateral microinjections of pentobarbital (200 μg/side) into the MPTA. This table shows results obtained form the pentobarbital microinjections. The asterisks (*) indicate statistically significant reduction in the electrophysiological parameter following pentobarbital microinjections compared to its respective baseline value (p < 0.05, Paired Student’s t test). Legend: SFR: spontaneous firing rate (spikes/s) FI: firing index Su‐ER magnitude: magnitude of sural nerve‐evoked STT response (number of spikes/trial) Su‐ER latency: mean latency of sural nerve‐evoked STT response (ms) Sc‐ER magnitude: magnitude of sciatic nerve‐evoked STT response (number of spikes/trial) Sc‐ER latency: mean latency of sciatic nerve‐evoked STT response (ms)  104  Results  Combined results of Groups II & III STT Neurons Electrophysiological Parameter  Baseline  SFR (n = 6)  15.4 ± 2.8  FI (n = 6)  89.3 ± 3.7  Su-ER Magnitude (n = 10) Su-ER Latency (n = 10) Sc-ER Magnitude (n = 10) Sc-ER Latency (n = 10)  minutes after pentobarbital (200 μg/side) microinjections 15 30  2  9.3 ± 1.7 15.4 ± 4.6 9.7 ± 1.8 43.6 ± 2.3  9.1 ± 3.2  *  56.1 ± 11.1 6.3 ± 1.7  *  *  15.3 ± 2.9 7.2 ± 1.9  *  44.4 ± 3.7  10.7 ± 3.3  *  58.4 ± 12.1 7 ± 1.7  15.4 ± 3.9  *  43.2 ± 3.2  12.8 ± 2.5  * 64.9 ± 12.7*  *  6.9 ± 1.7  60 10.8 ± 3.5 72.8 ± 8.5  *  9.7 ± 1.6  15.4 ± 4.5  15.9 ± 4  *  6.7 ± 1.8  42..4 ± 3.9  44.7 ± 3.6  7 ± 1.5  7.1 ± 1.7  Table 3.8 Summary of combined results of effects of bilateral microinjections of pentobarbital into the MPTA of the isoflurane‐anesthetized rat preparation on electrophysiological parameters of Group II and Group III STT neurons. The results are expressed as the group means (± SEM). The asterisks (*) indicate statistically significant reduction in the electrophysiological parameter following pentobarbital microinjections compared to its respective baseline value (p < 0.05, Paired Student’s t test).  Legend: SFR: spontaneous firing rate (spikes/s) FI: firing index Su‐ER magnitude: magnitude of sural nerve‐evoked STT response (number of spikes/trial) Su‐ER latency: mean latency of sural nerve‐evoked STT response (ms) Sc‐ER magnitude: magnitude of sciatic nerve‐evoked STT response (number of spikes/trial) Sc‐ER latency: mean latency of sciatic nerve‐evoked STT response (ms)  105  Results 3.3. RESULTS OF HISTOLOGY OF MICROINJECTION SITES  In twelve rats, microinjections of pontamine blue dye solution were carried out in the MPTA using the same stereotaxic coordinates that were used for PB/Vh microinjections. Figure 3.32 illustrates coronal sections of brainstems from three rats depicting the anatomical location of the MPTA site in the brain stem marked by either microinjections of pontamine blue dye (B and C) or cannula track mark (A).  The dye microinjections were found to be in brain areas corresponding to the following stereotaxic coordinates of Paxinos and Watson (2007): AP: ‐7.2 to ‐8 mm from bregma, ML: 1.1 to 1.2 from the midline and DV: ‐ 7.0 to ‐7.7 mm from the dorsal surface of the brain. The dye spread was found to be limited to a distance of 1.5‐2 mm in the mediolateral and dorsal‐ventral directions from the microinjection sites (Fig. 3.31 A, boxed area). This corresponds precisely to the location of MPTA as reported in the earlier studies (Devor and Zalkind, 2001; Sukhotinsky et al., 2005; Voss et al., 2005; Sukhotinsky et al., 2006; Sukhotinsky et al., 2007). Figure 3.33 summarizes the data of anatomical locations of dye marks in the MPTA obtained from twelve rats.  106  Results  Figure 3.32 Representative examples of anatomical location of MPTA in the rat brainstem. The figure shows coronal sections of brainstems of three different rats taken at the level of – 7.8 mm caudal to bregma. The section in A shows track mark (black rectangle) made by microinjection cannula directed toward the MPTA. The MPTA region marked by the dye (black rectangle) is depicted in section B. Figure C shows right coronal section of the brainstem with MPTA (square), which is magnified (10 X) in the right hand picture. In the  107  Results  Figure 3.32 continued magnified image, the cavity made by the tip of the microinjection cannula is marked by arrow. Note the dye spread around the cavity within the brain parenchyma. Legend: Aq – central aqueduct, DRD – dorsal part of dorsal raphe nucleus, MnR – median raphe nucleus, MPTA – mesopontine tegmental anesthesia area, PMnR – paramedian raphe nucleus, SPTg – subpeduncular tegmental nucleus  108  Results  Figure 3.33 Summary of anatomical locations of dye microinjections within the MPTA of rat brain stem. The locations of dye microinjections (black, filled circles) obtained from coronal sections of six rat brain stems are plotted on a representative section of brain stem obtained from the brain atlas of rat (Paxinos and Watson, 2007 electronic version) at the level of – 7.92 mm caudal to bregma. The green squares represent the locations of tract marks made by the tip of microinjection cannulae obtained from six other rat brain stems. The dye used was pontamine blue (2%) in 4M NaCl. The figure also shows abbreviated names of some important structures surrounding the MPTA. Legend: Aq – central aqueduct, DRD – dorsal part of dorsal raphe nucleus, DRV – ventral part of dorsal raphe nucleus, MnR – median raphe nucleus, MPTA – mesopontine tegmental anesthesia area, PMnR – paramedian raphe nucleus, PnO – oral part of pontine reticular nucleus, PTg – pedunculopontine tegmental nucleus, scp – superior cerebellar peduncle, SPTg – subpeduncular tegmental nucleus  109  Discussion  CHAPTER 4 DISCUSSION  4.1. Introductory Remarks  The recent suggestion that the mesopontine tegmental anesthesia area (MPTA) in the brainstem of the rat may function as an important node for the induction of general anesthesia has challenged the non‐specific theory of the general anesthetic actions within the CNS (Devor and Zalkind, 2001). The microinjections of GABAA –mimetic agents into this particular area of the rat brainstem induced classic, reversible general anesthesia‐like symptoms, namely, unconsciousness, analgesia, atonia and EEG synchronization (Devor and Zalkind, 2001). The observations by Devor and Zalkind (2001) were based on standardized behavioral test assessment techniques, which can often be misinterpreted or obscured by the inherent limitations, per se, in the techniques used and/or complex physiological phenomena occurring during the test state of anesthesia. For example, the analgesic effects of microinjections of pentobarbital (PB) as reported by Devor and Zalkind (2001) could be a manifestation of the animal’s inability to respond to the noxious stimuli due to the resulting muscle atonia. In other words, the animals may have been experiencing pain to the noxious stimuli but could not respond because of muscle paralysis caused by PB microinjections. Further, in behavioral testing paradigms, such as those employed by Devor and Zalkind (2001), discrimination between true analgesia, unconsciousness and anxiolysis is often ambiguous. Thus, the analgesia reported through the behavioral assessments can  110  Discussion not be unequivocally related to the “true analgesia”. Purported neurophysiological mechanisms of “true analgesia” are only conjectured at best. Therefore, it seemed appropriate to provide experimental evidence that would shed light on how analgesia arises by performing the electrophysiological studies presented in this thesis.  In this thesis, the focus was on analgesia, simply because one of the most important purposes of general anesthesia is to allow pain‐free surgery. Several lines of evidence suggest that the spinal cord is an important site of action for general anesthetic‐induced analgesia (de Jong et al., 1968; de Jong et al., 1969; de Jong et al., 1970; Heavner, 1975; Kitahata et al., 1975; Kullmann et al., 1989; Collins et al., 1995; Yamamori et al., 1995; Antognini, 1997; Antognini and Carstens, 1998). Further, intravenously injected thiopental, a barbiturate anesthetic, suppresses the spontaneous, peripheral nerve, and glutamate‐driven excitatory responses of spinal sensory neurons comprising the dorsal spinocerebellar tract (DSCT) and spinoreticular tract (SRT) in chronic, drug‐free cats (Soja et al., 2002). Thus, in the quest of exploring the physiological mechanism(s) for analgesia induced by PB microinjections into the MPTA, a logical site to target is the spinal cord.  The spinothalamic tract (STT) is considered to be the most important spinal ascending pathway in the mammals conveying nonnociceptive and nociceptive transmission to the brain (Willis, 1985; Willis and Coggeshall, 2004; Willis, 2007). This being so, it seemed logical to test whether the analgesia caused by the microinjections of PB into the MPTA, was due to the reduction in the sensory inflow via the STT. Accordingly, certain electrophysiological properties of the identified STT neurons in the lumbar spinal cord of  111  Discussion the isoflurane‐anesthetized rat preparation were studied before, during, and after microinjections of PB into the MPTA using extracellular recording techniques.  The principal hypothesis that was tested was that the bilateral microinjections of the barbiturate anesthetic, pentobarbital into the mesopontine tegmental anesthesia area (MPTA) of the isoflurane‐anesthetized rat suppress sensory inflow through the spinothalamic tract.  The following discussion focuses on: 1) technical considerations, 2) general properties of STT neurons, 3) inhibition of spontaneous and evoked responses of STT neurons after PB microinjections into the MPTA, 4) time course of PB actions on STT neurons 5) possible mechanism(s) of PB‐mediated suppression of STT neurons through the MPTA, and 6) possible role of MPTA in the control of pain transmission.  4.2. Technical Considerations: Animal Model, Study Design, Histology, and Statistical Analysis  The spinothalamic tract (STT) system in the acute rat preparation was chosen to study the effects of PB microinjections into the MPTA on sensory (nociceptive) transmission. The experiments were performed in the rat, as opposed to other species such as the cat, rabbit, guinea pig, etc., principally because the precise location of the MPTA region was well described in the rat brain (Devor and Zalkind, 2001; Voss et al., 2005).  112  Discussion A chronic, unanesthetized cat preparation (Soja et al., 1995) perhaps would have been more desirable and scientifically more appropriate in this study, because of the obvious non‐requirement for the use of the anesthetic needed to prepare the spinal cord for STT neuron recording as well as the lack of neuronal distortion caused by the anesthetic and surgery itself (Soja et al., 1995; Soja, 2007). In this unique preparation, STT neurons could have been recorded under “natural” conditions during the state of wakefulness akin to DSCT and SRT neurons (Soja et al., 2002; Soja, 2007). However, it is not presently known whether loci, that are functionally equivalent to the rat MPTA exist in chronic animal preparations, such as the cat, and, if they do, there is currently no data on their precise locations within the brain or their physiological function(s). Moreover, to our knowledge, no reliable chronic rat preparation is yet available for recording the activities of the lumbar sensory neurons.  Indeed a chronic rat model is difficult to prepare with current techniques because of the small size of the vertebrae and relative fragility of the vertebral column. Such factors preclude one from securing a permanent implant chamber for unit recording (Wall, 1967; Soja, 2007). Indeed, the development of a viable and reliable chronic animal preparation akin to the chronic cat preparation developed by Soja and colleagues (Soja et al., 1995; Soja, 2007) would require significant time and resources that are beyond the scope of this thesis.  Even with these limitations, the present study provided significant findings of STT neuron modulation mediated by PB through the MPTA. The results of the present study provide a provenance to warrant further studies of PB microinjections into the MPTA in the  113  Discussion chronic unanesthetized animal preparation that is free of neural distortion caused by preparative surgery and systemic anesthetic agents.  These caveats not withstanding, by necessity, the present study required the use of isoflurane, an inhalational general anesthetic, to produce a state of surgical anesthesia to prepare the animal for STT neuron recording and intracerebral microinjection procedures. In addition, pancuronium, a neuromuscular blocker, was used to block the movements induced by electrical stimulation of peripheral nerves. The abolition of movements produced by neuromuscular blockade is essential in studies of this kind, but it should be recognized that this benefit is offset by the pancuronium‐induced de‐afferentation of large‐ diameter muscle spindle afferents and their sensory afferent input to STT neurons (Foreman et al., 1979). Hence, the surgical preparation of the spinal cord together with differential effects of isoflurane on spinal neurons (Antognini and Carstens, 1999a, b; Antognini et al., 1999; Jinks et al., 1999; Jinks et al., 2003; Haseneder et al., 2004; Cuellar et al., 2005) may have altered the overall segmental and/or descending controls that impinge on STT neurons (Soja et al., 2002; Soja, 2007). Although the STT neurons examined in this thesis were, by necessity, recorded under “artificial” conditions (Soja, 2007) the actions of the barbiturate anesthetic PB, when microinjected into the MPTA, were nevertheless reproducible and interpretable.  One of the striking observations of this study was suppression of STT neurons by one general anesthetic (pentobarbital) in the presence of the other background anesthetic (isoflurane). Probably the most critical experimental caveat of this study was the presence of background anesthetic (isoflurane) that was continuously present throughout the  114  Discussion experimental protocol. Ethical constraints necessitated having the animal anesthetized at a surgical plane of anesthesia at all the times. Even though, the isoflurane levels were minimized as much as possible, while still maintaining a surgical plane of anesthesia, it cannot be ruled out that complex interactions of isoflurane and PB may have occured on the neural circuitry involved in the MPTA‐mediated STT neurons suppression and, for that matter, throughout other parts of the neuraxis including the spinal cord STT neurons being recorded. It is possible that the isoflurane used to anesthetize the animal may have already suppressed the excitability of STT neurons before PB microinjection and therefore contributed to an “occlusion” phenomenon. However, in the absence of “true baseline” of under anesthesia‐free control, further assessment of such interactions is impossible in conventional “acute” animal preparations. Nevertheless, one would expect an enhanced suppression of STT neurons in the absence of any background anesthetic. Such a possible scenario would be expected to occur only in a chronic rat preparation, which is currently not available (also, see Soja et al., 2001; Soja, 2007).  Standard techniques for antidromic identification were used to confirm that the recorded spinal neuron in this study projected to the thalamus. Such techniques have also been used previously for identifying STT neurons in the rat (Giesler et al., 1976; Menetrey et al., 1984; Palecek et al., 1992; Chen and Pan, 2002; Zhang and Giesler, 2005). All STT neurons reported in this study fulfilled the following three “gold standard” antidromic criteria: 1) a constant antidromic latency (< 0.2 ms), 2) high‐frequency following (> 300 Hz), and 3) a collision between the putative antidromically and orthodromically propagated action  115  Discussion potentials (Lipski, 1981; Ammons, 1989; Palecek et al., 1992; Soja et al., 1995). Thus, all the recorded spinal neurons reported in this thesis were confirmed to project to the contralateral thalamus and therefore were identified as STT neurons.  One of the formidable technical challenges of this thesis was to record from the same STT neuron for a protracted time wherein each STT neuron served as its own control before and after microinjections of vehicle control solution (Vh) or PB. Here, the basic electrophysiological parameters would be determined at regular intervals following microinjection of Vh into the MPTA followed by microinjection of PB with a recovery period between the two microinjections. This approach requires stable recording conditions that exceed four hours. Inevitably, during the course of this thesis, many STT neurons were inadvertently “lost” during the baseline data collection period or during microinjection procedure.  The microinjection procedure, itself increased the probability of losing the neuron due to minute vibrational disturbances to the experimental preparation, despite conscientious efforts to minimize such disturbances. For these reasons it was not possible to repeat the microinjections of either Vh or PB twice in the same neuron in every animal. As a consequence of these technical issues, the data collections occurred essentially randomly and “post hoc” data parsing allowed pooling into three distinct groups of STT neurons. These three groups of STT neurons, Group I (vehicle control‐only), Group II (pentobarbital‐ only) and Group III (vehicle control followed by pentobarbital group) were utilized to test the principle hypothesis that the bilateral microinjections of pentobarbital into the MPTA of  116  Discussion the rat brain suppress sensory inflow through the STT. In addition, the data grouping provided additional controls on STT neuron excitability as discussed later.  To verify that the PB/Vh were microinjected actually in the MPTA pontamine blue dye was microinjected at the end of all recording protocols. For this, the same volume of the dye, i.e., 1 μL, was injected into each side of the MPTA. The animal was then perfused 1 h after the dye microinjections. Thus, the same time period of 1 h was followed after dye microinjections as that followed after Vh/PB microinjections. An unavoidable technical caveat of this procedure was that the microinjection cannulae were required to be withdrawn from the brain for loading the pontamine blue dye into them. The dye‐filled cannulae were subsequently lowered into the brain at the stereotaxic coordinates previously used for microinjections of Vh/PB. Thus, the possibility can not be ruled out that the position of the dye‐filled cannulae in the MPTA was not exactly the same as that used for microinjections of PB/Vh.  However, several measures were taken to minimize the possible error occurring during repositioning of the dye‐filled cannulae into the microinjection site in the brain. First of all, the movement of the cannulae was kept limited only in the dorsal‐ventral direction. The anterior‐posterior and mediolateral positions of the cannulae were undisturbed. Secondly, before lowering the dye‐filled cannulae into the brain, the zero reference position for dorsal‐ventral movement, which was the dorsal surface of the brain, was always confirmed to be the same as that used for Vh/PB microinjections. Notwithstanding this technical caveat, as shown in the Results section, the histological examination of the brain  117  Discussion sections confirmed that the location of dye injections was indeed in the MPTA. This was confirmed from the description of the structures surrounding the MPTA (Devor and Zalkind, 2001) and stereotaxic atlas of the rat brain (Paxinos and Watson, 2007). In six rats, location of the microinjection site was confirmed by examining the location of the dye. In six other rats, the tips of tract marks made by microinjection cannulae were used to confirm the location of microinjection site within the MPTA.  In this thesis, two statistical tests were used to analyze the data obtained, namely, a paired Student’s t test and a repeated measures one‐way analysis of variance (ANOVA). Surprisingly, where the paired Student’s t test indicated significant differences, the repeated measures one‐way ANOVA failed to detect such differences. Two possible reasons that may explain this are: 1) the relatively small sample size and, 2) the variability of the data within the small sample size. However, since the principal concern was to detect significant differences in each STT neuron parameter post‐microinjection at each time point against the respective pre‐microinjection baselines rather than among the time points, a paired Student’s t test was considered more appropriate and was therefore used in the final data analyses.  The justification for using a paired Student’s t test for the final analyses was further corroborated by the professional advice sought through the Statistical Consultation Services provided by the Department of Statistics at the University of British Columbia. Thus, not only the techniques and parameters used to the test the hypothesis but also the choice of the most appropriate statistical test for detecting significant differences appears to be of  118  Discussion paramount importance in accepting or rejecting the null hypothesis that microinjection of PB into the MPTA has no effect on STT neuron excitability and ultimately, the interpretation of data obtained vis‐à‐vis anesthetic action of PB in the MPTA. This issue is of considerable scientific importance given the inherit ramifications related to warranting further studies on pentobarbital’s actions on STT neurons via the MPTA.  4.3. STT Neurons: General Properties  Spinothalamic tract (STT) neurons recorded in this thesis project were located at spinal cord depths ranging from 97 to 1528 μm with an average depth of 772 ± 397 μm below the surface of the spinal cord. This range corresponds to laminae I to V of the rat spinal grey matter (Molander et al., 1984; Grant and Koerber, 2004) as indicated by Palecek et al. (1992). The laminar organization of the lumbar spinal cord in the rat (Molander et al., 1984; Grant and Koerber, 2004) is closely similar to Rexed’s (1952) laminae in the cat spinal cord. The in situ recording depths were determined primarily from the electronic display of the hydraulic microdrive. The precise determination of the laminar locations of the STT neurons within the spinal cord by this technique can often be erroneous due to several factors, including, but not limited to, the inherent dead space in the hydraulic microdrive, constant presence of cerebrospinal fluid and/or mineral oil covering the exposed spinal segment that can obscure one from determining the “true” dorsal surface of the cord as zero reference for measuring the recording depth (Soja et al., 2001). Also, the relatively smaller size of the spinal cord of the rat in comparison to the other mammalian spinal cords, e.g. cat, can exaggerate such  119  Discussion errors to a greater extent. The only precise method to determine the depth of the recording is to employ intracellular horseradish peroxidase (HRP) labeling procedures which stain the entire soma‐dendritic processes of the recorded neurons (Brown et al., 1977). This technique, however, was far beyond the scope of the present thesis project. Nevertheless, the average depth of the STT neurons studied in this thesis project corroborates that reported by Palecek et al. (1992).  The mean antidromic latency of 18 STT neurons reported in this thesis measured 4.4 ± 0.6 ms (range: 3.7 – 5.8 ms) while the mean axonal conduction velocity was estimated to be 18.6 ± 2.5 m/s (range: 14.3 – 23 m/s). These values are in close agreement with those reported previously for the STT neurons in the lumbar spinal cord of the rat (Giesler et al., 1976; Menetrey et al., 1984; Palecek et al., 1992). The conduction velocities of lumbar STT neurons in the rat reported by Giesler et al. (1976) as well as Palecek et al. (1992) were in the range of 14 ‐ 26 m/s. Similarly, Menetrey et al. (1984) have reported a mean antidromic latency of 4.5 ± 0.5 ms with an estimated mean conduction velocity of 17.8 ± 2.0 m/s. Notably, the population of STT neurons recorded by Menetrey et al. (1984) was located in deeper laminae (VII, VIII and IX). On the other hand, most of the STT neurons reported in this thesis were located in laminae I to V, which corresponds more closely to findings reported by Giesler et al. (1976) and Palecek et al. (1992). Thus the STT neuron population included in this study, based on recording depth, seems to be similar to that reported by Giesler et al. (1976) and Palecek et al. (1992). In a more recent study, Zhang and Giesler (2005) have reported two populations of the STT neurons in the cervical enlargements of anesthetized rats. One  120  Discussion population of STT neurons located in the superficial dorsal horn had an estimated mean conduction velocity of 5.1 ± 0.4 m/s while the other, which was located in the deep dorsal horn, had an average conduction velocity of 8.9 ± 0.8 m/s. These conduction velocities are 2‐ 3 times slower than that reported (~ 19 ms/s) in this thesis. However, in the Zhang and Giesler (2005) study, the STT neurons were recorded from the cervical enlargement (C7 – C8) of the rat spinal cord, while the STT neurons in the present study were recorded from the L1 spinal segment. Moreover, the majority of antidromically recorded STT neurons in the Zhang and Giesler (2005) study projected to posterior thalamic nuclei rather than to the more rostrally located VPL nucleus. Thus, differences between the values of the latency, the conduction velocities as well as axon termination sites reported in this thesis and those by Zhang and Giesler (2005) are likely to be due to the different populations of the STT neurons across the various spinal cord segments. The relatively faster conduction velocities of the STT neurons in the present study suggest that their axons are of larger diameter, and are probably myelinated compared to those neurons reported by Zhang and Giesler (2005), which seem to be of smaller diameter and may be sparsely myelinated.  To determine whether STT neuron conduction velocity was related to the spinal recording depth, a correlation analysis was carried out between these two parameters. The analysis revealed no correlation between the axonal conduction velocity and the spinal recording depth of STT neurons. Similar observations have been reported for lumbar STT neurons in cat (Ammons, 1987) and monkey (Ammons, 1989) spinal cord. In contrast, Palecek et al. (1992) have reported that the more superficial STT neurons in the lumbar  121  Discussion spinal cord of the rat had slower conduction velocities. Similarly, Giesler et al. (1976) have noted that the STT neurons located in the superficial dorsal horn of the lumbar spinal cord of the rat had significantly slower conduction velocities than those located in the deep dorsal horn. Thus, while some lines of evidence suggest a correlation between the recording depth and conduction velocity of STT neurons, the present study revealed no such correlation. This may be due to the relatively small sample size or segmental level of the spinal cord where STT neurons were recorded. Moreover, greater dorsal‐ventral laminar spread of STT neurons reported in the present thesis project may have diluted a possible correlation between these two parameters. Had many more STT neurons been examined, such a correlation may have manifested itself. The significance of such a correlation in the absence of any procedural test is not clear.  4.4. Inhibition of Spontaneous and Evoked Responses of STT Neurons by Pentobarbital Microinjections into the MPTA  The results of the present study clearly show that the cellular excitability of STT neurons was significantly and reversibly suppressed following pentobarbital (PB) microinjections into the MPTA. Given that the vehicle control solution, containing ethanol, a CNS depressant itself, did not alter these parameters after microinjections into the MPTA indicates that the effects were due to the barbiturate anesthetic PB itself. The observation that the PB microinjections into the MPTA suppress the animal responses to noxious stimuli (Devor and Zalkind, 2001; Voss et al., 2005) can be explained on the findings from this study.  122  Discussion That is, the “analgesia” observed in the Devor and Zalkind (2001) study can be attributed, in part, to a reduction in sensory inflow through the STT. In the following sub‐sections, the effect of intracerebrally microinjected PB on each of the parameters is discussed. The possible neurophysiological mechanisms including possible neural interconnections between the MPTA neurons and the pain transmission and modulation systems within the brain and spinal cord are also discussed.  4.4.1. Spontaneous Firing Rate  All of the L1 STT neurons recorded in the isoflurane‐anesthetized rat preparation displayed ongoing spontaneous activity. The ongoing activity of the STT neurons at the resting state could be due to the input from central and peripheral neurons as well as the intrinsic integrative properties of the STT neurons (Surmeier et al., 1989; Zhang et al., 1991a). STT neurons receive tactile and nociceptive afferent input via Aβ, Aδ and C fibers (Willis, 1985; Willis and Coggeshall, 2004). In addition, STT neurons recorded in the lumbosacral and thoracolumbar segments of monkey spinal cord are known to receive proprioceptive (Foreman et al., 1979) and enteroceptive inputs (Milne et al., 1981), respectively. Tissue damage resulting from surgical manipulations can serve as sufficient stimuli to activate primary afferents, thereby providing tonic excitatory input to the STT neurons. The spontaneous spike activity under the present experimental conditions is also offset by the depressant actions of isoflurane required to maintain a surgical plane of anesthesia (see Vahle‐Hinz and Detsch, 2002). Despite these considerations, the mean baseline spontaneous  123  Discussion firing rate (SFR) of the 18 STT neurons measured ~13 spikes/s. This SFR is much higher than that of the STT neurons recorded in the lower lumbar (L4 ‐ L5) spinal cord segments reported in previous studies (Palecek et al., 1992; Chen and Pan, 2002). In the studies reported by Palecek et al. (1992) as well as Chen and Pan (2002), the SFR measured approximately 5 spikes/s and < 1 spikes/s, respectively. The differences between the background activities of the STT neurons reported herein and those by Chen and Pan (2002) as well as Palecek et al. (1992) may be due to a number of factors, including, but not limited to, the type of anesthetic used. In the later two studies PB was used as the general anesthetic agent, whereas in the present study, isoflurane was used to induce a surgical plane of anesthesia.  A recent study from our laboratory has shown that the lumbar STT neurons recorded in the same isoflurane‐anesthetized rat preparation, as used in this thesis, are under descending modulatory influences, which are dependent on the type of general anesthetic used (Soja et al. unpublished observation). STT neurons in the dorsal horn are largely under tonic descending facilitatory influences when recorded during isoflurane anesthesia. The descending influences on the same recorded STT neurons undergo a switch from facilitatory to inhibitory influences during the changeover from isoflurane to PB anesthesia (Soja et al. unpublished observation). Thus, the relatively higher baseline spontaneous activity of the STT neurons reported here compared to previous studies might be due in part to the use of isoflurane anesthesia. There appears to be no other studies on STT neuron responses recorded in the rat under isoflurane anesthesia in the literature that  124  Discussion can corroborate these observations. This is rather surprising given the widespread use of isoflurane as an inhalational anesthetic in human surgical procedures (Evers et al., 2006).  The mean baseline SFR of the STT neurons in Groups I, II, and III were ~ 9, 12, and 19 spikes/s, respectively. The mean baseline SFR of the Group III STT neurons seemed to be somewhat higher than that of Groups I and II. However, statistical comparison between baseline SFR of the three groups showed no significant difference. The apparently different baseline SFR of the STT neurons in these three groups is likely due to the sampling bias leading to preferential selection of anatomically different subsets of STT neurons among the groups (Surmeier et al., 1989). Moreover, lamina‐specific differences in the spontaneous discharge properties of dorsal horn neurons in the in vitro rat spinal cord preparation have been reported recently (Ruscheweyh and Sandkuhler, 2002). Hence, to rule out the possibility of sampling bias, a correlation analysis between the recording depth of the STT neurons below the surface of the spinal cord and their respective baseline SFR within each group was performed. The correlation analysis revealed no significant relationship existed between the recording depth and SFR in any of the three groups (see Results 3.2.2.1). Thus, the apparent differences in the SFR of the STT neurons in the three groups were not likely due to sampling bias. Taken together, the statistical analysis indicates that all the STT neurons recorded in this thesis represent a homogenous population of STT neurons. In contrast to the results of this study, a significant relationship has been reported between the recording depth and SFR of STT neurons recorded in the primate lumbosacral spinal cord (Surmeier et al., 1989). The relatively higher firing rates of STT neurons located in the deeper  125  Discussion laminae (IV‐VI), as reported by Surmeier et al. (1989) have been attributed to the large dendritic trees of these neurons, and subsequently, large number of afferent connectivities (Surmeier et al., 1989).  In this study, slight variations were found in the background spike activities of individual STT neurons following microinjections of vehicle control solution (Vh) into the MPTA. For some STT neurons, the spike activity was slightly reduced, for others there was slight increase in the spike activity. Collectively, when the entire population of STT neurons was considered, spike activity did not differ significantly following microinjections of pentobarbital (PB)‐free vehicle into the MPTA. This provided a reliable control for the actions of PB on STT neuron activity. Hence the actions of PB on spontaneous spike activity could be reasonably attributed to the actions of PB itself. These observations correspond to the behavioral observations in conscious rats reported by Devor and Zalkind (2001), where microinjections of control vehicle into the MPTA failed to induce general anesthesia‐like state and the associated analgesia.  As shown in the Results section, microinjections of PB into the MPTA resulted in a rapid and significant decrease in the mean SFR of the STT neurons. In the case of the Group II STT neurons, which were treated only with the PB microinjections, this suppression was marked and measured > 50%. Similarly, a > 45% decrease was observed in the average SFR of the Group III STT neurons. In the case of both of these neuron groups, this suppression in the SFR was rapid and occurred within two minutes of PB microinjections. The rapid change in the SFR strongly suggests a local action(s) of PB in the MPTA. In the case of the  126  Discussion Group II STT neurons, which were treated only with PB microinjections, this suppression in the mean SFR was sustained for fifteen minutes and was followed by recovery toward the baseline firing rate within thirty minutes. The SFR of Group III STT neurons was suppressed for a shorter time period (compared to the Group II STT neurons) and recovered to the baseline firing rate within fifteen minutes of the PB microinjections.  Interestingly, in the case of the Group III STT neurons, after recovery within fifteen minutes of PB microinjections, there was an “anomalous” but significant decrease in the SFR after sixty minutes of the PB microinjections. This decrease was more pronounced than that observed immediately following the PB microinjections. It is not known presently, whether this second anomalous decrease in the SFR was due to a drug effect at the level of MPTA neurons or due to some other factor(s).  There are however two possible explanations for this anomalous decrease in the SFR. First, there is a possible depression of spontaneous spike rate through the re‐distribution of PB to the spinal cord through the CSF and/or blood flow, where it may have directly suppressed the activities of the STT neurons. However, this possibility seems to be less likely, since this second drop in the SFR after initial recovery was not observed in case of Group II STT neurons, which were recorded for the same length of time after PB microinjections. The second possibility is related to the physiological state of the spinal cord. Group III STT neurons were tested first with microinjections of Vh before PB microinjections. For this group, the spinal cord was exposed for over 10 h (including the time required for post‐laminectomy surgical procedures, antidromic identification of STT  127  Discussion neuron, baseline recording, and recordings following Vh microinjections, recovery period, and recording following PB microinjections). Such a long exposure time may have induced time‐dependent deterioration in the physiological state of the spinal cord, despite considerable conscientious efforts to maintain the animal’s vital signs and depth of anesthesia within the constraint of normal physiological limits. Irrespective of the mechanism underlying this decrease in the spike activity of Group III STT neurons at the 60 min post PB‐microinjection time point, the actions of PB in the MPTA on STT neurons are consistent with the same time course as that observed by Devor and Zalkind (2001) who first showed the anesthetic actions of PB in the MPTA.  4.4.2. Interspike Interval Parameters  Many CNS neurons undergo alterations in their spike firing patterns when shifts occur in the animal’s behavioral state as reflected by predictable changes in their ISIH distributions as well as CV and CD values. For example, thalamocortical (TC) neurons operate in two modes depending on the animal’s behavioral state. During wakefulness, the spike discharge of TC neurons was characterized by a regular tonic firing pattern that is referred to as “relay mode” (Baker, 1971; Steriade and McCarley, 2005). When the animal’s behavioral state shifts from wakefulness to non‐rapid eye movement (NREM) sleep accompanied by EEG spindle oscillations, TC neurons switch from relay mode to a “bursting or oscillatory mode” that is characterized by high‐frequency bursts of spikes with long interspike intervals. Clear cut changes occur in TC neuron ISIH distributions, which, in turn, reflect spike burst activity that are further confirmed by changes in CV and CD (Steriade and McCarley, 2005).  128  Discussion Changes in the spike firing pattern have also been shown to occur in some ascending spinal sensory neurons, including the DSCT and SRT neurons during sleep vs. wakefulness (see Soja et al., 2007; Soja et al., 1996). Since sleep and general anesthesia share many common characteristics, it was logical to examine in the present study if microinjections of PB into the MPTA altered the firing patterns of STT neurons. To determine whether microinjections of PB into the MPTA altered the spike pattern of STT neurons, the interspike interval histogram (ISIH) distributions as well as the values for coefficient of variation (CV) and coefficient of dispersion (CD) derived from 2 min spike trains were analyzed. As shown in the Results section, microinjections of neither Vh nor PB altered any of the interspike interval parameters, indicating that the spike patterns of STT neurons as a population did not change. Had STT spike patterns changed from a regular “relay mode” to a “burst mode” following PB microinjections, such a finding would suggest that PB may exert effects on neural networks in the brain affecting sensory neurons in the spinal cord that overlap with those of the TC axis. Further, if the STT spike pattern had shown “burst‐like” activity following PB microinjections into the MPTA, such a finding could also possibly suggest that common neural networks mediate components of the states of natural sleep and MPTA‐ mediated general anesthesia.  4.4.3. Antidromic Firing Index  The antidromic firing index (FI), which indicates the probability of the STT neurons responding to stimuli applied to the thalamus, was used to indirectly measure the excitability of the STT neurons. The FI method has long being used for testing changes in the  129  Discussion postsynaptic excitability of motor neurons (Hunt, 1955; Lloyd and McIntyre, 1955; Wilson and Burgess, 1962), group Ia and Ib afferents (Wall, 1958; Willis et al., 1976), tooth pulp afferents (Lisney, 1979; Cairns et al., 1996) and some descending fiber systems (Rudomin and Jankowska, 1981). In studies of primary afferent terminals, per se, the presynaptic inhibitory changes in the terminal excitability are proposed to be related to a process of primary afferent depolarization (PAD) or primary afferent hyperpolarization (PAH) mediated through the axoaxonic synapses impinging on the primary afferent fibers (Rudomin, 1999; Rudomin and Schmidt, 1999). In case of spinal motoneurons, a decrease in the antidromic FI can be attributed to hyperpolarization of the motoneuron cell body (Lipski, 1981). A decrease in the FI could also be due to disfacilitation that can be the result of increased outwardly directed potassium conductances that might occur when supraspinal (monoaminergic) influences are dampened (Wilson and Burgess, 1962; Zhang et al., 1991b, a). An advantage of this technique is that changes in the cellular excitability can be measured even if the neuron does not have any spontaneous or background activity (Lipski, 1981).  As shown in the Results section, the FI of STT neurons were not significantly altered by microinjections of Vh into the MPTA. Indeed the FI was the most stable of the three electrophysiological parameters measured following Vh microinjections since it was intentionally set to ~ 90% by manually adjusting the intensity of thalamic stimulation.  The FI was significantly suppressed following PB microinjections. In the case of Group II STT neurons, the suppression was observed within two minutes, while in Group  130  Discussion III STT neurons, the suppression was observed 15 min after PB microinjections. The FI recovered within 30‐60 min of the microinjections. Interestingly, in the case of one STT neuron (STT neuron # 17), the FI was abolished within two min of the microinjections. In the same neuron, the spontaneous firing rate was suppressed by ~ 82% within 2 min of PB microinjections. An abolition of FI might suggest a nonspecific block of axonal conduction due to local anesthetic‐like activity of PB (Blaustein, 1968; Staiman and Seeman, 1974). This scenario might occur if PB diffused to the more lateral sites in the brain stem where the STT axons pass. It has been shown that the spread of a 1 μL volume of solution within the brain parenchyma is limited to the volume of ~ 1 mm3 surrounding the microinjection site (Lomax, 1966; Myers, 1966). In rat, the area in the brainstem through which the STT axons pass along with the lateral lemniscus is more than 2 mm farther from the MPTA (Willis, 1985; Paxinos and Watson, 2007). Thus, in case of the present series of experiments, the possibility of local anesthetic‐like actions of PB occurring due to the diffusional spread to the STT axons passing through the brainstem seems less likely. Further, the abolition of FI following microinjections of PB into the MPTA was observed for only one STT neuron. Therefore PB‐mediated abolition of FI in this case may be due to some other mechanism.  Changes in the FI could also be due to several factors, besides the drug that include but not are limited to, movement of the spinal cord with relation to the recording electrode, changes in the electrode resistance, temperature oscillations, and fluctuations in the excitability if smaller number of trials used to determine the FI (Willis et al., 1976; Duenas and Rudomin, 1988; Cairns et al., 1996). In the present study, conscientious efforts were  131  Discussion made to minimize the influence of these factors and to ensure that the changes in the FI were due to PB. In such experiments, the main source of cord movement is respiration. This was minimized by proper adjustment of the spinal clamps (see Materials and Methods, section 2.1.6.). To eliminate fluctuations in the electrode resistance, tungsten electrodes were used in this thesis, which have a stable resistance as opposed to glass microelectrodes. Furthermore, the body temperature of the animals was always maintained within the physiological limits throughout the experiment (see Materials and Methods). Moreover, we used a sufficient number (75) of antidromic trials to compute the FI, which is higher than the small number of trials (~10) used in the previous studies (Willis et al., 1976). Finally, the stable baseline FI after microinjections of Vh indicates minimal influence of these “extraneous factors” on the FI. Thus FI was a relatively stable parameter of STT neuron excitability.  In conclusion, the suppression of STT neuron FI by microinjection of PB into the MPTA indicates a reduced excitability of the STT neuron, presumably due to somatic membrane hyperpolarization and/or increased conductance (Whitehorn and Burgess, 1973; Willis et al., 1976; Lipski, 1981). This change in the STT neuron membrane polarization may likely have been due to an action of PB at the level of MPTA via descending influences impinging on the recorded neurons.  4.4.4. Peripheral Nerve‐Evoked STT Responses In this thesis, the synaptic activation of STT neurons by sural and sciatic nerves resulted in a complex spike response. The evoked response consisted of a short presynaptic afferent  132  Discussion volley, followed by compound action potential and in most cases a burst of STT neuron spikes. In agreement with the SFR and FI, the mean response magnitude of synaptically‐ evoked responses of STT neurons to both the sciatic and sural nerve stimulation were significantly suppressed following PB microinjections into the MPTA. This suppression however, was observed in the case of STT neurons that were treated only with PB (i.e., Group II STT neurons).  Surprisingly, we did not see any significant changes in the mean magnitudes of both the sciatic as well as sural nerve‐evoked responses of Group III STT neurons after PB microinjections. The reason for this apparent discrepancy is presently unclear, although there was a trend towards suppression of the evoked responses. Here, the small sample size could be a principal contributing factor.  For three STT neurons, stimulation of sciatic nerve evoked short‐latency “early” responses and long‐latency “late” responses (see Fig. 3.8). Taepavarapruk et al. (2004) have reported similar type of peripheral nerve‐evoked responses of DSCT neurons in the lumbar spinal cord of cat. In the study of Taepavarapruk et al. (2004) the “early” and “late” responses are thought to be evoked via monosynaptic and polysynaptic linkages, respectively. Similarly, two types of burst discharges of STT neurons, evoked by sural nerve stimulation were observed by Beall et al. (1977) in the monkey spinal cord. Here, these early and late discharges are thought to be related to volleys in Aβ and Aδ fibers, respectively. It should however, be noted that as discussed further, the intensity used for the peripheral nerve stimulation was not enough to activate C fibers. Hence, it is less likely that “late”  133  Discussion component of the evoked responses herein was due to C fiber stimulation. Since, the “early” and “late” components of sciatic nerve‐evoked responses were observed in only three STT neurons no systematic analysis of the “early” and “late” evoked responses was performed.  The inhibition of peripheral nerve‐evoked responses could result from postsynaptic inhibition of the STT neuron or presynaptic inhibition of the primary afferent neurons. In this study, since PB was microinjected into supraspinal sites, the most probable source of inhibition of synaptic responses is through descending inhibition. A possible mechanism for this inhibition is discussed in detail in the further sections.  High‐frequency electrical stimulation at C‐fiber threshold intensities is known to induce long‐term potentiation (LTP) or long‐tem depression (LTD) of dorsal horn sensory neurons (Liu and Sandkuhler, 1995; Liu and Sandkuhler, 1997; Ikeda et al., 2000) and primary afferents (Randic et al., 1993). The stimulation of the sciatic and sural nerves at C fiber threshold intensities could have induced complex interactions in the dorsal horn through LTP/LTD, making interpretation of the results more difficult. For this reason, in the present study low‐intensity stimuli (two times the threshold intensity) were used for sciatic and sural nerve activation that were insufficient to activate C fibers. Thus the afferent nerve stimulation protocol at the intensities used in this study can by no means reflect noxious input to the STT neurons. Even though the responses of STT neurons to the noxious input were not studied in this thesis, the general suppression of STT neuron excitability would nevertheless be expected to reduce nociceptive transmission at the spinal level.  134  Discussion A second effect that could obscure the effect of PB on peripheral nerve‐evoked responses is the phenomenon of habituation. Repetitive application of peripheral stimuli can lead to suppression of spinal neurons, by a process called habituation (Griffin and Pearson, 1967; Macdonald and Pearson, 1979). In the present study, no habituation of evoked responses was observed after stimulation of either sciatic or sural nerves. This provided a stable baseline for both the sural and sciatic nerve‐evoked responses for comparison with the post‐microinjection responses.  In addition to the assessment of post‐synaptic responses of STT neurons, analyses of effects of PB microinjections into the MPTA on presynaptic input via primary afferents was also performed. Here, peak‐trough amplitudes and latencies of the presynaptic afferent volleys evoked by stimulation of the sciatic and sural nerves were compared before and after microinjections. As indicated in the Results section, the size of the presynaptic afferent volley evoked by both the nerves did not change significantly following microinjections of either PB or Vh. This data suggests that synchronous presynaptic input to the STT neurons provided by electrical stimulation of peripheral nerve trunks essentially remained unchanged after microinjections of PB into the MPTA. Thus, the suppression of peripheral nerve‐evoked STT neurons responses following PB microinjections into the MPTA may involve a post‐synaptic mechanism. Although, this type of analysis suggests that the same numbers of axons are activated following sciatic and sural nerve stimulation, it does not rule out a PB‐mediated decrease in the transmitter release from central terminal arbors of  135  Discussion primary afferent fibers in the spinal cord as a consequence of enhanced impulse traffic through axoaxonic synapses (Rudomin, 1999; Rudomin and Schmidt, 1999).  4.5. Time Course of Effects of Pentobarbital on the Spike Activity of STT Neurons  In this study the temporal profile of the STT neuron responses were also recorded after single bilateral PB microinjections. The results show that the spontaneous spike activity as well as orthodromic and antidromic evoked responses of the STT neurons were suppressed within 2 min of PB microinjections. This suppression was reversible and recovery occurred within 15‐30 min of microinjections. A similar time course of suppression of identified DSCT and SRT neurons (Soja et al., 2002) as well as unidentified dorsal horn sensory neurons (Nagase et al., 1994) in the cat spinal cord has been reported after intravenous injection of barbiturate anesthetics. In the behavioral studies of Devor and Zalkind (2001) as well as Voss et al. (2005), the anesthesia‐like state was induced within 1‐5 minutes of PB microinjections into the MPTA. The animals recovered from anesthesia within 40‐120 min of microinjections. In the present studies, the recovery of STT neurons following suppression was relatively shorter (~30 min) than that reported in the behavioral studies.  In summary, the overall time course of the suppression and subsequent recovery of spontaneous firing rate, antidromic firing index, and peripheral nerve‐evoked responses of STT neurons following microinjections of PB into the MPTA reported in this thesis overlap remarkably with the behavioral effects reported by Devor and Zalkind (2001). The data reported in this thesis provide a neurophysiological mechanism of the analgesia that is  136  Discussion associated with the general anesthesia‐like state after PB microinjections into the MPTA (Devor and Zalkind, 2001).  4.6. Possible Neural Mechanism(s) of Pentobarbital‐Mediated Suppression of STT Neurons through the MPTA  The results of the present series of experiments show that the microinjections of PB into the MPTA of the rat suppress both the spontaneous as well as the evoked orthodromic and antidromic activities of the STT neurons. Suppression of sensory inflow through the STT, which is considered to be the most important spinal nociceptive pathway, could be the possible physiological mechanism for the analgesia that accompanies the general anesthesia as reported by Devor and Zalkind (2001). Whether this suppression was at the post‐synaptic level or presynaptic level or both, is currently unknown. One way to determine whether a post‐synaptic mechanism is involved or not, is by using the extracellular recording technique in conjunction with the juxtacellular application of glutamate by either microiontophoresis (Willcockson et al., 1984) or microdialysis (Dougherty et al., 1992) technique at the site of the STT neuron under study. Glutamate is an excitatory amino acid neurotransmitter known to excite nearly all CNS neurons (Davies et al., 1979; Sahai, 1990; Greenamyre and Porter, 1994), including STT neurons (Willcockson et al., 1984; Dougherty et al., 1992). If microinjections of PB into the MPTA were found to suppress the glutamate‐ evoked STT responses, such a finding would be further indicative of post‐synaptic hyperpolarization. Intracellular recording paradigms designed to estimate STT neuron  137  Discussion conductance (Zhang et al., 1991a) can also be employed to confirm direct somatic inhibition following PB microinjections. The hyperpolarization could be the consequence of at least two neural processes, namely, direct inhibition via chloride‐gated conductances mediated via GABA or glycine and/or disfacilitation. The latter disfacilitation process could arise due to a possible increase in outward potassium conductance in the STT neuron membrane resulting from the withdrawal of supraspinal influences (Zhang et al., 1991b, a).  Two  possible  scenarios  are  presented  below  that  may  involve  somatic  hyperpolarization and/or disfacilitation of STT neurons following microinjections of PB into the MPTA. The first possibility is that PB microinjections into the MPTA may engage (in)directly endogenous descending pain control systems. Histochemical studies show that the neurons in the MPTA send their axonal projections to various regions in the brain and spinal cord which are involved in the control of pain transmission and modulation (Sukhotinsky et al., 2003; Sukhotinsky et al., 2006; Reiner et al., 2007). These sites in the brain include relay nuclei in the rostral ventromedial medulla (RVM), especially the nucleus reticularis gigantocellularis (NRGC) and nucleus raphe magnus (NRM) as well as the periaqueductal grey (PAG) and locus ceruleus (LC). The PAG exerts robust descending inhibition on the spinal pain transmission neurons including STT neurons through the relay centers in the RVM (Gerhart et al., 1984; Zhang et al., 1991b; Stamford, 1995; Bajic and Proudfit, 1999; Mason, 1999; Odeh and Antal, 2001; Millan, 2002; Fields et al., 2006). The MPTA neurons also send direct axonal projections to the spinal cord dorsal horn (Sukhotinsky et al., 2006). Thus, MPTA neurons are thought to modulate the activities of  138  Discussion these structures in the CNS. The microinjection of PB into the MPTA may act as an inhibitory switch through which GABAAergic mechanisms, in turn, might modulate various descending control mechanisms, mediated by NRGC, NRM, LC, and PAG, that ultimately lead to oligosynpatic suppression STT neurons excitability. Here again at the level of the STT neuron, the suppression may be due to direct classical, chloride mediated, postsynaptic inhibition, and/or disfacilitation mediated by outward potassium or other conductances (Zhang et al., 1991b, a).  A second scenario might include direct actions of PB microinjected into the MPTA on the STT neurons themselves. It is possible that intracerebrally microinjected PB may have reached the STT soma in the spinal cord by re‐distribution through the CSF and/or blood. The present thesis did not investigate this possibility. One method to test the re‐distribution of PB to the spinal cord is by blocking the nerve transmission in the spinal cord between the microinjection and recording sites, and recording the spike activity around PB microinjections. Spinal transmission can be blocked by reversible cold block spinalization (Wall, 1967; Soja and Sinclair, 1983). If the microinjections of PB into the MPTA suppress the STT neurons when spinal cord is in the reversible cold block state, then this effect can be attributed to a re‐distribution of PB to the spinal cord. In this thesis the suppression of STT spike activity was rapid; observed in < 2 min of the microinjections. This line of evidence further indicates actions of PB on local neurons in the site of microinjection that ultimately influence the STT responses in the spinal cord, rather than a re‐distribution phenomenon.  139  Discussion It is also possible that through the diffusional spread within the brain parenchyma PB may have reached the STT axons passing through the brainstem. In most of the mammalian species, including the rat, the STT axons pass in the brainstem in conjunction with the lateral lemniscus (Willis, 1985). Further, the average area of diffusional spread of 1 μL volume of solution microinjected into the brain is ~ 1 mm3 surrounding the microinjection site (Lomax, 1966; Myers, 1966). As mentioned previously, the area in the brainstem through which the STT axons pass along with the lateral lemniscus is >2 mm farther from the MPTA (Willis, 1985; Paxinos and Watson, 2007). Thus, the possibility that suppression of STT neurons occurred through diffusional spread of PB to the STT axons passing through the brain stem seems rather remote, and if so, the concentration of PB would have to be great enough to exert a local anesthetic action on the membranes of STT axons. This was further confirmed by the histological examination of the brain after microinjections of pontamine dye into the MPTA. The lateral spread of the dye was always found to be limited within 2 mm of the microinjection site.  Based on the published reports of the neuroanatomy of MPTA neurons and their projections within the CNS, as well as the electrophysiological findings of the present study, the possible neural mechanism for PB‐induced suppression of STT neurons is presented below. To aid in explaining the possible neural mechanism, hypothetical connectivities of MPTA neurons with the pain transmission and intrinsic control system(s) within the CNS are depicted in the Figure 4.1.  140  Discussion The MPTA contains GABAergic neurons as well as neurons bearing GABAA‐ receptors (Sukhotinsky et al., 2003). These neurons project to several key areas in the brain as well as to the spinal cord, which play important functions in pain modulation and transmission, respectively. The most prominent projections of the MPTA neurons to the descending pain control centers in the brain are: the periaqueductal grey (PAG), rostral ventromedial medulla (RVM), locus ceruleus (LC), and dorsal raphe nuclei (Sukhotinsky et al., 2003; Sukhotinsky et al., 2006; Reiner et al., 2007). In addition, the spinal cord dorsal horn receives distinct, direct projections from the MPTA (Sukhotinsky et al., 2006; Reiner et al., 2007). However, the synaptic interconnectivities of these projections with the sensory neurons in the dorsal horn are not yet clear. It is possible that the spinal projections of MPTA neurons may make direct synapses with the sensory tract neurons, such as STT neurons. This may provide an anatomical substrate for direct postsynaptic inhibition of STT neurons following PB microinjections into the MPTA. It is also possible the MPTA neurons may make direct axoaxonal connections with the central terminals of primary afferent neurons or indirect synapses via various interneurons. This would provide an anatomical substrate for primary afferent depolarization (PAD)‐like presynaptic inhibition (Rudomin, 1999; Rudomin and Schmidt, 1999) of STT neurons following PB microinjections into the MPTA. Again, it should be emphasized that currently no evidence exists on whether spinally projecting MPTA axons form axoaxonic synapses with primary afferent neurons or postsynaptic synapses with projection neurons in the spinal cord (Sukhotinsky et al., 2005; Sukhotinsky et al., 2006).  141  Discussion The PAG is one of the major sources of descending inhibition of spinal nociceptive transmission. The neurons in the PAG exert a disfacilitatory influence through descending projections that synapse directly onto the neurons in the RVM (Shah and Dostrovsky, 1980; Bajic and Proudfit, 1999; Mason, 1999; Odeh and Antal, 2001; Fields et al., 2006). The relay centers in the RVM, and the adjacent reticular formation, especially the nucleus reticularis gigantocellularis (NRGC) and nucleus raphe magnus (NRM), in turn, suppress the activities of spinal sensory neurons, including STT neurons (McCreery and Bloedel, 1975) through their serotonergic (Bowker et al., 1981; Liu et al., 2002; Millan, 2002; Fields et al., 2006) and/or GABAergic (Antal et al., 1996) projections to the dorsal horn. As stated previously, MPTA neurons also send axonal projections to the LC, which is one of the major sources of noradrenergic descending inhibition of spinal pain transmission (Basbaum and Fields, 1978; Millan, 2002; Fields et al., 2006). Spinally projecting neurons comprising the NRM and/or LC may also suppress the STT neurons through presynaptic inhibition of the primary afferents (Willis et al., 1977; Martin et al., 1979). Another potential mechanism underlying the suppression of STT responses to afferent nerve stimulation in this study may be due to the NRM and/or LC‐mediated PAD‐like presynaptic inhibition of primary afferent neurons (Willis et al., 1977; Martin et al., 1979; Rudomin, 1999; Rudomin and Schmidt, 1999).  Under resting conditions, the MPTA neurons may be spontaneously active and exert tonic inhibitory influences on the neurons in the PAG as well as on the relay centers in the RVM probably through GABAergic mechanisms (Sukhotinsky et al., 2003; Sukhotinsky et al., 2005). The possible sources of the tonic activity of MPTA neurons may be the intrinsic  142  Discussion properties of these neurons and/or the undefined projections from other brain areas to the MPTA neurons and/or the local interneurons. In addition, the spinally projecting neurons in the MPTA may themselves exert tonic descending facilitation of STT neurons by acting directly on the STT neurons and/or by suppressing local inhibitory interneurons and/or activating the excitatory interneurons which make synaptic connections with the STT neurons.  Alternatively, the spinally projecting neurons from the MPTA could exert descending inhibition of the ascending spinal sensory neurons, but this inhibition may be dampened by the inhibitory interneurons at the resting state (Sukhotinsky et al., 2005). The overall actions of the MPTA neurons on the pain modulatory centers in the brain as well as the dorsal horn sensory neurons may facilitate pain transmission through the spinal cord at the resting state.  Microinjections of PB and other GABAA receptor‐active anesthetics into the MPTA may suppress the tonic activity of the MPTA neurons (Devor and Zalkind, 2001; Sukhotinsky et al., 2003; Sukhotinsky et al., 2005). As a consequence, the tonic inhibitory influences on the PAG as well as the relay centers in the RVM and LC might then be removed. The disinhibited PAG neurons would then further excite the serotonergic and noradrenergic neurons of the in the RVM and LC, respectively, which, in turn, suppress the transmission through the STT. Further, the putative facilitatory input to the spinal sensory (STT) neurons through the direct spinal MPTA projections could be dampened by the  143  Discussion microinjections of PB. However, it should be noted that none of these scenarios has been addressed in the scientific literature and are therefore, speculative in nature.  144  Discussion  MIDBRAIN  Thalamus  PAG  SPINAL CORD  GABA?  RVM/LC  GABA/GLY?  MPTA  GABA/GLY?  MEDULLA  PONS  GABA?  5-HT, NE  RVM/LC  5-HT, NE  GABA/GLY  1˚ Afferent  STT Neuron  1˚ Afferent  Figure 4.1 Schematic of the hypothetical neural circuitry involved in suppression of STT neuron by pentobarbital microinjections into the rat MPTA. The MPTA neurons project to the descending pain control centers in the brain such as PAG, RVM and LC as well as to the dorsal horn of the spinal cord. The MPTA neurons may tonically suppress the descending  145  Discussion  (Fig. 4.1 continued) control centers at the baseline resting stage. Additionally, the descending projections of the MPTA to the spinal cord dorsal horn may facilitate pain transmission through STT via either a direct projection to the STT soma and/or indirectly through activation of excitatory interneurons and/or suppression of inhibitory interneuron(s) making synaptic connections with the STT neurons. The possible neurotransmitters involved in the MPTA actions include GABA and/or glycine. The descending pain control system suppresses pain transmission by direct actions on the STT through inhibitory interneuron(s) and/or indirectly by presynaptic inhibition of primary afferent neurons. The possible neurotransmitters involved include 5‐ HT, NE, GABA and/or glycine. Microinjections of pentobarbital into the MPTA may suppress the activity of MPTA neurons, probably via GABAAergic mechanisms, which in turn, would disinhibit the descending pain control centers in the supraspinal cites. The disinhibited pain control system, in turn, suppresses pain transmission through the STT.  Legend: PAG – Periaqueductal grey, RVM – rostral ventromedial medulla, LC – locus ceruleus, 5‐HT – serotonin, NE‐ norepinephrine, GLY – glycine, \ ‐ inhibitory neurotransmission,  ‐ excitatory neurotransmission  146  Discussion  4.7. MPTA: A Possible Pronociceptive Center in the Brain?  The neural connectivities of MPTA neurons with the endogenous pain modulation structures in the brain as well as the spinal cord (Sukhotinsky et al., 2006) suggest that MPTA may be playing important role in the descending pain modulation. Microinjections of barbiturate anesthetic, pentobarbital (PB) are hypothesized to inhibit the tonic activity of MPTA neurons (Devor and Zalkind, 2001; Sukhotinsky et al., 2005; Sukhotinsky et al., 2006). The results of the present study indicate that inhibition of MPTA neurons by PB suppress the spike activities of the STT neurons. This raises the question whether MPTA neurons overall, provide a facilitatory input to the spinal sensory neurons under physiological conditions. In other words, is MPTA a possible pronociceptive center in the brain?  Research over the past 10 years provides compelling evidence for the existence of descending pain facilitatory systems in the brain. The medulla contains several structures that have been shown to possess pronociceptive functions. Almeida and colleagues have reported that the medullary dorsal reticular nucleus (DRt) is a distinct source of pronociceptive input to the spinal cord dorsal horn (see review by Lima and Almeida, 2002). Stimulation of DRt neurons by glutamate decreases the tail‐flick latency (Almeida et al., 1996) while chemical and electrical lesions of the DRt suppress pain behavior (Almeida et al., 1999). Similarly, stimulation of DRt by glutamate showed enhanced responses of unidentified spinal nociceptive neurons to peripheral nerve stimulation at C‐fiber intensities, which were suppressed by application of lidocaine in the DRt (Dugast et al., 2003). The DRt neurons have been shown to be reciprocally connected with the RVM and  147  Discussion LC (Lima and Almeida, 2002). It would be interesting to know whether any connectivity exists between the MPTA and the DRt. Sukhotinsky et al. (2006) have not reported on such connections between the MPTA and DRt neurons. If such connections do exist, a potential modulatory influence of MPTA on DRt may exist that facilitates spinal nociceptive transmission.  The rostral ventromedial medulla (RVM), which contains the raphe nuclei and NRGC, have been shown to exert biphasic (antinociceptive and pronociceptive) modulation of the spinal nociceptive transmission (Zhuo and Gebhart, 1990, 1991, 1992; Urban and Gebhart, 1997; Zhuo and Gebhart, 1997; Zhuo et al., 2002). Electrical stimulation of the RVM induced a discharge pattern in the STT neurons, which was similar to that produced by the noxious stimulation of the skin (Giesler et al., 1981b). Similarly, occasional excitatory effects of electrical stimulation of the PAG and the reticular formation on identified spinomesencephalic tract (SMT) neurons have been observed in the cat (Yezierski, 1990). Fields and colleagues have shown that the RVM has three distinct neuronal populations: the ON‐cells, the OFF‐cells and the neutral cells (Fields et al., 1983a; Fields et al., 1983b). The ON‐ cells show a burst of their spike activity while the OFF‐cells show a pause in their activity, immediately before a noxious stimulus‐induced motor reflex. The neutral cells are not affected during the noxious stimulus‐induced reflex. It has been proposed that, activation of RVM ON‐cells permits or facilitates nociceptive transmission through the spinal cord while the activation of RVM OFF‐cells suppresses it (Fields et al., 1991). The role of RVM OFF‐cells  148  Discussion in descending inhibition of pain transmission has been confirmed from several other studies (Tortorici and Vanegas, 1994, 1995; Jones, 1996).  Given the direct synaptic inputs from the MPTA to the RVM neurons (Sukhotinsky et al., 2006) there may be possible MPTA inputs to the RVM ON‐ and OFF‐cells. Histological and/or physiological studies are required to confirm this possibility. However, if such connectivities are assumed then it may be possible that under resting conditions the MPTA neurons may be tonically activating the RVM ON‐cells (possibly through glutamatergic mechanisms)  and/or  inhibit  the  OFF‐cells  (probably  through  GABA/glycinergic  mechanisms), thus facilitating the spinal nociceptive transmission. At the same time, given the reciprocal connectivities of RVM and LC with the MPTA as well as the DRt neurons, the MPTA neurons may provide indirect tonic facilitatory influences to the DRt neurons through the RVM and LC. Inhibition of MPTA neurons by PB may remove the inhibitory influences on the RVM OFF‐cells and/or excitatory influences on the ON‐cells. The disinhibited RVM OFF‐cells in turn would suppress the nociceptive transmission through the spinal cord.  In summary, the results of this study along with the evidence from the literature suggest that MPTA may have a pronociceptive role along with other pronociceptive centers in the brain. Alternatively, it may be acting as the source providing pronociceptive drives to the other brain centers to facilitate spinal nociceptive transmission. Further studies are necessary to explore this possibility.  149  Discussion  4.8. Future Directions  The physiological role of MPTA in pain transmission and analgesia is not completely understood yet. Extensive neural connectivities of the MPTA with various structures in the brain as well as the spinal cord suggest that MPTA neurons may play an important role in sleep‐wake cycles as well as sensory‐motor integration (Sukhotinsky et al., 2003; Sukhotinsky et al., 2005; Sukhotinsky et al., 2006; Reiner et al., 2007; Sukhotinsky et al., 2007). The reciprocal connections of MPTA neurons with the various structures of the endogenous pain control system suggest that the MPTA may play important role in the descending pain modulation, probably in a feedback/feed forward fashion. The results of the present study indirectly suggest the possible descending pronociceptive influences of MPTA on the spinal nociceptive transmission. These descending facilitatory effects may be mediated through the direct projections of the MPTA neurons to the spinal cord and/or indirectly through the medullary relay centers. Further studies could be designed to explore these possibilities.  The STT pathway, by no means, is the sole spinal nociceptive pathway. The effects of PB microinjection into the MPTA on other groups of ascending spinal nociceptive neurons including, the spinoreticular tract (SRT), spinomesencephalic tract, spinohypothalamic tract, and spinocervicothalamic tract are unknown. Therefore, the analgesia observed during the general anesthesia‐like state after PB microinjections cannot be completely explained on the basis of STT suppression alone. However, as indicated earlier, systemic administration of thiopental has been shown to suppress the spontaneous as well as evoked responses of the identified SRT neurons, which like STT neurons, do convey nociceptive input to the brain  150  Discussion (Soja et al., 2002). It is, therefore plausible that the microinjection of PB into the MPTA may suppress the excitability of other spinal nociceptive tract systems. This study thus provides a plausible physiological mechanism for the analgesia induced by PB microinjections into the MPTA. The findings of this study provide a foundation for further physiological studies to elucidate the neural networks involved in MPTA‐mediated general anesthesia. The results of this thesis provide more questions in light of the preceding discussion than the original hypothesis addressed. A myriad of experimental paradigms including in vivo and in vitro techniques can be used to dissect out the neural circuitry underlying the analgesia (not to mention the atonia, amnesia and unconsciousness) that accompanies the state of general anesthesia induced by PB in the MPTA. Future studies that might be carried out are briefly discussed below.  1. The MPTA is only a functionally defined region in the brain. The anatomical boundaries as well as the intrinsic properties of neurons comprising the MPTA are not presently known. This warrants further studies along this direction.  2. As stated earlier, the disadvantages of using acute animal preparation require the development of a reliable chronic animal preparation for such studies. The small size of vertebrae and relatively fragile vertebral column make the rat a technically difficult preparation for chronic recording studies in the spinal cord. A chronic cat preparation (Soja et al., 1995) might be more suitable for physiological studies of MPTA neurons under “near natural” conditions. However, the presence of neurons, functionally equivalent to the rat MPTA in the cat is yet unknown. Hence,  151  Discussion exploratory studies are required to identify such functional area(s) in the cat brain. Since the chronic cat preparation has been successfully used for sleep‐wake studies, it could be ideal to compare the effect of sleep states on STT neuron activity before induction of the anesthetic state by PB microinjections into the MPTA in this preparation. These cat preparations could be used to investigate behavioral state‐ related changes in MPTA neurons as well as and the effects of systemic vs. focal administration of PB into the MPTA.  3. Suppression of STT neuron activity following microinjections of PB into the MPTA is hypothesized to be due to the descending inhibition mediated via the direct projections of MPTA neurons to the dorsal horn and/or indirect pathway through the endogenous descending control system. The synaptic connectivities of the direct MPTA projections with the spinal cord dorsal horn as well as the neurotransmitter system(s) involved are presently unknown. On the other hand, the endogenous descending pain control system, which comprises of relay nuclei RVM and LC form direct synaptic connectivities or indirect synaptic connectivities through the interneurons with the sensory tract cells in the spinal cord (Stamford, 1995; Millan, 2002; Fields et al., 2006). Further, the descending projections of RVM and LC use serotonin (5‐HT) and norepinephrine (NE) as the neurotransmitters, respectively at the spinal cord level (Basbaum and Fields, 1978; Mason, 1999; Millan, 2002; Fields et al., 2006). To assess if suppression of STT neurons following PB microinjections into the MPTA involves activation of indirect descending inhibitory pathways, changes in the 5‐HT and NE levels at spinal site of STT cell body can be analyzed using  152  Discussion microdialysis procedures. In this case, measurement of neurotransmitter release by microdialysis and extracellular recording of STT neurons can be simultaneously performed before and following microinjections of PB into the MPTA (Sorkin et al., 1988). The changes in the levels of 5‐HT and NE following PB microinjections can be measured by subjecting the collected microdialysis fluid to the high performance liquid chromatographic (HPLC) analysis. Following PB microinjections into the MPTA, if there is suppression of STT neurons with corresponding increase in the 5‐ HT and/or NE levels, it would indicate activation of indirect descending inhibitory pathway(s). This procedure, however, can not be used to assess the involvement of direct projections of MPTA neurons as the neurotransmitter systems as well as synaptic connectivities in the spinal cord are not yet known.  4. Reciprocal projections of MPTA neurons with various structures involved in the control of sleep‐wake cycles (Sukhotinsky et al., 2007) suggest that MPTA neurons may play important role in the regulation of sleep‐wake cycles as well as modulation/control of sensory transmission through the spinal cord during sleep‐ wake cycles. Since the earlier reports of Carli and colleagues (Carli et al., 1967a; Carli et al., 1967b) compelling evidence now indicates that the sensory transmission through the spinal cord is under modulatory influences during sleep (see, Soja, 2007). Along these lines, previous studies from our laboratory in chronic cat preparation have shown that the sensory transmission through various identified spinal ascending pathways, including the DSCT, SRT and trigeminothalamic tract undergo sleep‐wake cycles‐dependent modulatory changes (Soja et al., 1993; Cairns  153  Discussion et al., 1995; Soja et al., 1996; Soja et al., 2001; Taepavarapruk et al., 2002; Taepavarapruk et al., 2004). It may be conceivable that STT neurons also undergo modulation in their activities during sleep‐wake cycles. Thus, it may be feasible to study the spike activities of MPTA neurons in conjunction with the identified spinal sensory pathways such as STT neurons across the sleep‐wake cycles. If a functionally equivalent MPTA locus is present and identified in the cat, a chronic cat preparation (Soja et al., 1993) can be conveniently used for such inquiries. Such studies may provide novel information on the actions of MPTA neurons on spinal sensory transmission during natural sleep‐wake cycles. Several in vivo pharmacological experiments can also be carried out using the aforementioned paradigms.  5. MPTA neurons are reported to send axonal projections to the endogenous motor control systems as well as the ventral horn (Sukhotinsky et al., 2005). This provides an opportunity to study whether the motor neurons are suppressed after PB microinjections, which may provide potential mechanism for the atonia. This could also be performed reliably in a chronic cat preparation and the anesthetic actions of PB into the MPTA on motor outflow could also be compared with that, which occurs during naturally occurring rapid eye movement (REM) sleep (Chase et al., 1989; Soja et al., 1991).  154  Summary and Conclusions  CHAPTER 5 SUMMARY AND CONCLUSIONS 1. Extracellular spike activity of antidromically identified spinothalamic tract (STT) neurons in the L1 spinal segment was studied following bilateral microinjections of pentobarbital (PB, 200 μg/side) into the mesopontine tegmental anesthesia area (MPTA) in the brainstem of isoflurane‐anesthetized rat preparation. 2. The group mean spontaneous firing rate (SFR) of STT neurons was significantly suppressed by 45‐50% following microinjections of PB into the MPTA. This suppression of the SFR returned to pre‐injection baseline values within 15‐30 min of PB microinjections. The firing pattern of STT neurons was not altered by microinjections of Vh or PB. The group mean antidromic firing index (FI), along with the magnitudes of sural, as well as sciatic nerve‐evoked orthodromic responses of STT neurons were also significantly  and  reversibly  suppressed  following  PB  microinjections.  Thus,  microinjections of PB into the MPTA suppressed both the spontaneous as well as antidromically and orthodromically‐evoked spike activities of STT neurons. 3. There were no changes in the group mean peak‐trough amplitude of the presynaptic afferent nerve volley following PB microinjections. This suggests that the suppression of STT neuron excitability by PB microinjections into the MPTA is probably a post‐synaptic phenomenon on the neurons under study and/or a reduction in transmitter release via presynaptic inhibition.  155  Summary and Conclusions 4. The data of the present thesis project provides evidence that the MPTA is a barbiturate‐ sensitive area in the rat brain stem that is capable of diminishing sensory inflow through the STT. 5. In summary, the physiological basis for the behavioral analgesia, which accompanies the reversible general anesthesia‐like state after bilateral microinjections of PB into the MPTA, can be partially attributed to the suppression of STT neurons, which transmit nociceptive signals to the brain. The suppression of STT neurons following PB microinjections into the MPTA may be achieved directly through the axonal projections of MPTA neurons to the spinal cord dorsal horn and/or indirectly through the projections of MPTA neurons to the descending pain control system(s) in the brainstem and at the level of the STT neuron itself. Such actions of PB may involve pre and/or postsynaptic forms of inhibition.  156  References  REFERENCES Aimone LD, Gebhart GF (1986) Stimulation‐produced spinal inhibition from the midbrain in the rat is mediated by an excitatory amino acid neurotransmitter in the medial medulla. J Neurosci 6(6): 1803‐1813.  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