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Species and developmental differences in mammalian respiratory rhythm generation Gajda, Barbara Marie 2007

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SPECIES A N D DEVELOPMENTAL DIFFERENCES IN MAMMALIAN RESPIRATORY RHYTHM GENERATION  by BARBARA MARIE GAJDA B.ScH., The University of Winnipeg, 2004  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in  THE FACULTY OF GRADUATE STUDIES  (Zoology)  THE UNIVERSITY OF BRITISH COLUMBIA September 2007  © Barbara Marie Gajda, 2007  Abstract Mammalian neonates can recover spontaneously from hypothermia-induced respiratory arrest when re-warmed (termed autoresuscitation). As a rat ages, autoresuscitation ability is lost during a transitional period ('developmental window') between 16 - 20 post-natal days (PND) so that hypothermic respiratory arrest results in death for a mature rat. Hamsters retain the ability to autoresuscitate past this developmental window. The retention of this ability in hamsters implies that there may be fundamental differences in the central rhythm generator (CRG) of rats and hamsters. This study tests the hypothesis that the contribution to respiratory rhythm generation of the putatively rhythmogenic persistent Na current (I ) and Ca *-activated non-selective cation +  2  NAP  current (IQAN). two currents which may facilitate the initiation of breathing after arrest, is different between rats and hamsters. Because autoresuscitation ability is lost during development, we also test the hypothesis that the I  NAP  and I  CAN  contribution to respiratory rhythm generation change as  a rat ages. We applied riluzole (I  NAP  blocker) and flufenamic acid (FFA;  blocker) to the arterially  perfused in situ working heart-brainstem preparation in hamsters and two age groups of rats (12 14 PND, >23 PND). Application of riluzole and FFA to rats and hamsters showed that elimination of I  NAP  and I  CAN  resulted in profound decrease in phrenic burst frequency in hamsters  with little change in rats. This result is consistent with the hypothesis that a phylogenetic difference exists in the mechanism of setting respiratory rhythm in the C R G of rats and hamsters. Comparisons between young and weaned rats showed that young rats tended to be more sensitive to the application of riluzole and FFA than weaned rats. The small differences observed between young and weaned rats in the reliance on I  NAP  and  for respiratory rhythm generation are  consistent with the hypothesis that a developmental change occurs in the C R G of rats during maturation. Increasing the proportion of C 0 that the preparations were exposed to increased 2  neural ventilation in weaned rats suggesting that I  NaP  and ICA provide a source of excitatory drive N  to the CRG.  ii  Table of contents Abstract  ii  Table of contents  iii  List of tables  v  List of figures  vi  List of abbreviations  viii  Acknowledgements  ix  1. Introduction  1  1.1.  Hypothermic respiratory arrest and autoresuscitation  1  1.1.1. Background information on respiratory rhythm generation 1.2. Phylogeny and respiratory rhythm generation 1.2.1. Do phylogenetic differences exist in the roles of I and ICAN in respiratory rhythm generation? 1.3. Ontogeny and respiratory rhythm generation 1.3.1. Do ontogenetic differences exist in the roles of I and I in respiratory rhythm generation?  1, 4  NaP  NaP  4 5  CAN  5  2. Methods  8  2.1. 2.2. 2.3.  8 9 10  In situ working heart-brainstem preparation Experimental protocol Data and statistical analysis  '.  3. Results  16  3.1. Riluzole application 3.2. Flufenamic acid application 3.3. Riluzole and FFA coapplication 3.3.1. Coapplication at 5% C O 3.3.2. Coapplication at 7% C 0 z  :  2  4. Discussion 4.1. 4.2. 4.2.1. 4.2.2. I  16 18 19 20 21 40  Technical considerations 40 Phylogenetic differences in I and ICAN contribution to rhythm generation 42 Hamsters required I to set respiratory rhythm 42 Respiratory burst amplitude is affected in rats more than hamsters by the elimination of NaP  NaP  and ICAN 4.2.3. Hamster neural ventilation decreases more than rat upon elimination of I and ICAN46 4.2.4. Increasing drive only rescues 'breathing' in rats 47 4.3. Ontogenetic differences in I and I N 8 4.3.1. Respiratory rhythm in young rats is more sensitive to application riluzole and FFA.... 48 4.3.2. Young rats more dependent on I and ICAN weaned rats for producing motor output 50 4.3.3. Ventilation in young rats declined more than in weaned rats 51 4  4  NaP  NaP  4  NaP  CA  m  a  n  NaP  iii  4.3.4. Increasing drive only rescues 'breathing' in weaned, not young, rats 4.4. Speculations 4.5. Conclusions  51 53 54  5. References  55  6. Appendix  61  List of tables Table 2-1: The range, mean and median perfusate flow rate values to in situ preparation  12  Table 2-2: The range, mean and median weight values of young rats, weaned rats and weaned hamsters used in the in situ preparation  12  Table 3-1: Mean and the range of baseline phrenic nerve burst frequency for young rats, weaned rats and weaned hamsters. The values are ± standard error 24 Table 3-2: The number of animals with phrenic nerve activity at indicated concentrations of riluzole  24  Table 3-3: The number of animals with phrenic nerve activity at indicated concentrations of FFA. 28 Table 3-4: Number of preparations producing respiratory rhythm during coapplication of riluzole and FFA 32 Table 3-5: Number of preparations exposed to 7% C 0 coapplication of riluzole and FFA  2  producing respiratory rhythm during 37  Table 3-6: Alterations in phrenic nerve burst frequency, T[ and T of young rat, weaned rat and weaned hamster preparations exposed to 5% C 0 or 7% C 0 . Values are expressed as means±standard error. N=number of preparations 37 E  2  2  Table 6-1: T, and T values for young rats, weaned rats and weaned hamsters in the riluzole trials. Asterisk (*) denotes significance from baseline 61 E  Table 6-2: T, and T values for young rats, weaned fats and weaned hamsters in the FFA trials. ..61 E  Table 6-3: T[ and T data for young rats, weaned rats and weaned hamsters in the trials with coapplication of riluzole and FFA  62  Table 64: T[ and T values for young rats, weaned rats and weaned hamsters in the trials with coapplication of riluzole and FFA  62  E  E  v  List of figures Figure 1-1: Respiratory column located in the brainstem of mammals  7  Figure 1-2: Bursting in respiratory neurons  7  Figure 2-1: Schematic diagram of the in situ working heart-brainstem preparation  13  Figure 2-2: Approximate location of decerebration in a sagittal section of the rat brain  13  Figure 2-3: Representative neurograms of respiratory activity in young rat and hamster in situ preparations  14  Figure 2-4: Variables measured from a phrenic neurogram  15  Figure 3-1: Thirty second representative raw (PHR) and rectified integrated (JTHR) neurograms of PHR nerve activity in response to increasing concentrations of riluzole 25 Figure 3-2: The change in phrenic nerve burst frequency (A), amplitude (B), and neural ventilation (nV , C), corrected for vehicle controls, in response to increasing concentrations of riluzole 26 E  Figure 3-3: Pyramid plots for young rats (A), weaned rats (B) and weaned hamsters (C) for increasing concentrations of riluzole  27  Figure 3-4: Thirty second representative raw (PHR) and rectified integrated (JTHR) neurograms of PHR nerve activity in response to increasing concentrations of FFA 29 Figure 3-5: The change in phrenic nerve burst frequency (A), amplitude (B), and neural ventilation (nV , C), corrected for vehicle controls, in response to increasing concentrations of FFA 30 E  Figure 3-6: Pyramid plots for young rats (A), weaned rats (B) and weaned hamsters (C) for increasing concentrations of FFA  31  Figure 3-7: Frequency, amplitude and neural ventilation (nV ) all declined as FFA concentration increased in weaned rat preparations (n=6) 32 E  Figure 3-8: Thirty second representative raw (PHR) and rectified integrated (JTHR) neurograms of PHR nerve activity in response to increasing concentrations of riluzole + FFA 33 Figure 3-9: The change in phrenic nerve burst frequency (A) and amplitude (B), and neural ventilation (nV , C) corrected for vehicle controls, in response to increasing concentrations of riluzole + FFA 34 E  Figure 3-10: Expected values for neural ventilation (nV ) based on additive action of riluzole and FFA compared to the actual nV for young rats (A), weaned rats (B) and weaned hamsters (C) 35 E  E  vi  Figure 3-11: Pyramid plots for young rats (A), weaned rats (B) and weaned hamsters (C) for increasing concentrations of riluzole and FFA combination  36  Figure 3-12: Changes in frequency, corrected for vehicle controls, for young rat (A), weaned rat (B) and hamster (C) preparations exposed to perfusate equilibrated with 5% C O or 7% C 0 and coapplication of riluzole and F F A 38 z  2  Figure 3-13: Changes in amplitude, corrected for vehicle controls, for young rat (A), weaned rat (B) and hamster (C) preparations exposed to perfusate equilibrated with 5% C 0 or 7% C 0 and coapplication of riluzole and F F A 38 2  2  Figure 3-14: Changes in neural ventilation (nV ), corrected for vehicle controls, for young rat (A), weaned rat (B) and hamster (C) preparations exposed to perfusate equilibrated with 5% C 0 or 7% C O and coapplication of riluzole and F F A 39 E  2  z  vii  List of abbreviations  BotC  Botzinger Complex  CRG  Central rhythm generator  CVN  Cervical vagus nerve  cVRG  Caudal ventral respiratory group  FFA  Flufenamic acid  I  Ca -activated non-selective cation current 2+  CAN  IK+-LEAK  K leak current  I  NaP  Persistent Na current  I  NaT  Transient Na current  +  +  +  mV  Millivolts  YI V  E  Neural ventilation  pA/pF  Unit for current density (picoamperes/picoFarads)  pgQ  preBotzinger Complex  pj^pj)  Post-natal days  pjL  Riluzole  rVRG  Rostral ventral respiratory group  y  Expiratory duration or interburst interval  y  Inspiratory duration  \r  Membrane potential  yp^Q  Ventral respiratory group Hypoglossal nerve  viii  Acknowledgements This research project would not have been completed if not for the technical expertise and friendship of Angelina Fong who originally taught the working heart-brainstem preparation to me in Missouri and after continued to be a source of advice and encouragement with respect to the many downs and ups the W H B P as well as the experimental design of this project. I would like to also acknowledge my supervisor, Bill Milsom, for his expertise and support and for deliberately creating a familial environment in our lab. Thank you to my committee members Vanessa A u l d and Matt Ramer for their helpful feedback on experimental design, data analysis and thesis drafts. Thank you also to Jeffrey T. Potts at the University of MissouriColumbia for hosting me in his lab for two weeks in February 2005 while I learned the W H B P and for loaning me a pump that I have yet to return. I would like to thank Bruce Gillespie, Vince Grant, and D o n Brandys in the mechanical and electrical shops for building and repairing many essential parts of my rig. I would like to extend my gratitude and affection to past and present members of the Milsom lab who encouraged my academic growth as well as opportunities for fun and silliness throughout my degree. Finally, I would like to recognize my parents, Krys and Bo, brother Marek, my mentors Ted and Nancy, fellow members of Development and Peace and my friends and community at St. Mark's Chapel/St. Ignatius Parish whose prayers and encouragement have sustained me over the past three years. I especially would like to remember Fr. Brian Burns who passed away suddenly in June 2007 because of his formative impact on my life.  ix  1. Introduction 1.1. Hypothermic respiratory arrest and autoresuscitation When the body temperature of an animal is reduced in an uncontrolled manner (hypothermia), respiration first slows then stops (termed hypothermic respiratory arrest). Most adult mammals can withstand only a certain level and duration of hypothermia before vital processes fail irreversibly. Respiratory arrest is followed by cardiac arrest and eventually death (Tattersall & Milsom, 2003). Neonatal mammals, however, have been observed to remain in a state of respiratory arrest for up to three hours at hypothermic temperatures and fully recover (Adolph, 1951) by spontaneous resuscitation (autoresuscitation) upon gradual re-warming without the need for artificial intervention (as is necessary for adults) (Adolph, 1951; Hill, 2000). Marshall (2005, unpublished) determined that a critical period in development exists when maturing rats lose the ability to autoresuscitate. This 'developmental window' begins at 14 post-natal days (PND) and autoresuscitation ability is completely lost by 18 - 20 P N D ; after this window hypothermic respiratory arrest results in death in the absence of intervention (Marshall, 2005). Hamsters, permissive hibernators, also have autoresuscitation ability; however they maintain this ability past the developmental window of rats (Marshall, 2005) and throughout adulthood (Andrade et al, unpublished). Therefore, hamsters do not exhibit a developmental window as rats do. Hypothermic respiratory arrest occurs due to the failure of the respiratory central rhythm generator (CRG) in the medulla to generate a rhythm, rather than the failure of motoneurons to propagate the signal or the loss of motor function in the respiratory muscles (Mellen et al, 2002; Tattersall & Milsom, 2003). Therefore, a difference may exist in the C R G between species that maintain autoresuscitation ability (hamsters) compared to species that do not (rats).  1.1.1. Background information on respiratory rhythm generation The motion of breathing is comprised of two mechanical movements: inspiration and expiration. However from the perspective of neurobiology, breathing is comprised of three neural phases: inspiratory, post-inspiratory and expiratory phases. The post-inspiratory phase, arising between the antagonistic inspiratory and expiratory phases of muscle activity, is associated with 1  passive expiration and constriction of upper airway muscles (Richter, 1982). Respiratory neurons responsible for generating respiratory rhythm are classified according to the neural phase in which the neurons display activity and are found within a large heterogeneous group of respiratory neurons in the ventrolateral medulla known as the ventral respiratory group (VRG) (Figure 1-1). The V R G extends from the first cervical segment (CI) rostrally to abut the facial nucleus (Ellenberger 6k Feldman, 1990). It can be subdivided rostrocaudally into 4 divisions: the Botzinger Complex (BotC), the preBotzinger Complex (PBC), the rostral V R G (rVRG) and the caudal V R G (cVRG). The P B C has been postulated to be the site of inspiratory rhythm generation in the brainstem. The P B C can be isolated chemically or physically from the rest of the respiratory column in vitro and will continue to generate respiratory-like rhythm (Smith et al, 1991)(Johnson et al, 2001). Several ablation studies in intact animals (rats and goats) have shown that destroying PBC neurons will severely disrupt breathing (Wenninger et al, 2004)(Gray et al, 2001)(McKay et al, 2005), supporting the hypothesis that the P B C is vital for respiratory rhythm generation. Because respiratory rhythm can be maintained when inhibitory synaptic connections are blocked, inspiratory neurons with intrinsic bursting (pacemaker) ability expressing a voltagedependent persistent sodium current (I ) were proposed to be necessary to set respiratory NaP  rhythm (Smith et al, 1991; Johnson et al, 1994; Del Negro et al, 2002a; Del Negro et al, 2002b). I  NaP  is active at more negative membrane potentials than other currents: I  NaP  is active between -60  and -40 mV, transient sodium current (I ) is active at -34 m V (Richter & Spyer, 2001)(Butera NaX  Jr. et al, 1999)(Urbani 6k Belluzzi, 2000)(Alzheimer et al, 1993). I  NaP  is found in several neural  tissues (such as the rostral ventrolateral medulla, spinal cord, petrosal ganglion, suprachiasmatic nucleus, neocortex) (Rybak et al,  2003)(Urbani 6k Belluzzi, 2000; Darbon et al,  2004;  Kononenko et al, 2004; Faustino 6k Donnelly, 2006) and is expressed in all respiratory neurons in the r V R G , however at a greater current density (pA/pF) in P B C interneurons (Ptak et al, 2005). I  NaP  facilitates respiratory rhythm generation by promoting plateau potentials  and  repetitive firing and by increasing neuron excitability (as depicted in Figure 1-2). I  NaP  -60 m V and depolarizes the membrane to the activation threshold for I  (Figure 1-2BCD),  NaX  is activated at  2  leading to voltage-dependent bursting (Figure 1-2B©) (Feldman 6k Del Negro, 2006). During the action potential I  NAT  becomes inactivated rapidly; however, due to the slow inactivation of I  NAP  , the  membrane potential remains elevated allowing repetitive bursting (Figure 1-2B) to continue until I  NAP  is eventually inactivated (Figure 1-2BCD) (Crill, 1996)(Del Negro et al, 2002b)(Feldman & Del  Negro, 2006). By not rapidly inactivating following an action potential, I  NAP  prevents the  afterhyperpolarization that follows every spike thereby promoting another burst in the spike train (Figure 1-2A©) (Lee & Heckman, 2001). I  NAP  is expressed ubiquitously in the PBC; however not all PBC neurons have bursting  pacemaker properties even though I  NAP  appears to perform similar functions in non-pacemaker  and pacemaker neurons. Therefore, bursting pacemaker neurons are characterized not just by the presence of I  NAP  but by a ratio of I  to K leak currents (I . K) +  NAP  K+  a n o <  LEA  by the ability to continue  firing when synaptically isolated (Del Negro et al, 2002b; Ptak et al, 2005). I  NAP  confers bursting  properties and increases the excitability of all respiratory neurons (pacemaker and nonpacemaker). However, by interacting with I . EAK. ^ a P may have a more specific role in setting K+ L  inspiratory rhythm in bursting pacemaker neurons with intrinsic rhythmicity by increasing the rate of membrane depolarization to threshold levels for action potential generation (-40 mV) (Figure 1-2B©) (Del Negro et al, 2002b). Another current thought to bestow bursting pacemaker properties to another set of inspiratory neurons within the PBC is the calcium-activated non-selective cation current, I Q ^ (Thoby-Brisson 6k Ramirez, 2001; Pena et al, 2004). ICAN is found in many regions of the brain and body (Gogelein et al, 1990; Hall & Delaney, 2002)(Cho et al, 2003) including the V R G (Pena et al, 2004; Del Negro et al, 2005). ICA is expressed in nearly all PBC neurons; its N  mechanism is voltage-independent but requires increased cytoplasmic Ca in order to be activated 2+  (Del Negro et al, 2005)(Hall 6k Delaney, 2002). Upon activation by increased intracellular Ca , z+  ICAN produces a depolarizing current (Partridge et al, 1994). Since the  channels do not rapidly  inactivate, ICAN contributes to bursting and plateau potentials also (Partridge et al, 1994)(Hall 6k Delaney, 2002)(Rekling 6k Feldman, 1997). Recent evidence also has suggested that I  CAN  contributes to the inspiratory drive potential which is a 10 - 30 mV depolarization of the  3  membrane that brings the neuron to threshold potential for bursting (Figure 1-2A) thereby increasing the excitability of respiratory neurons (Pace et al, 2007a). Thus, both I  and I  NAP  CAN  are involved in generating respiratory output by promoting  bursting, producing plateau potentials and increasing neuronal excitability. Their proposed role in setting respiratory rhythm is derived from the observation of respiratory neurons in the PBC that continue bursting when synaptically isolated (Thoby-Brisson 6k Ramirez, 2001; Pena et al, 2004). These neurons have been presumed to be rhythmogenic because of their intrinsic bursting ability (conferred by I  NAP  or I AN) and are proposed to compose a kernel of respiratory neurons, C  embedded in a network of reciprocally inhibited respiratory neurons, that sets the baseline rhythm for the respiratory network and eventually produces rhythmic motor output (Smith et al, 2000). Riluzole, a neuroprotective and antiepileptic drug, blocks I assess the importance of I  NAP  NAP  and has been widely used to  to respiratory rhythm generation (Thoby-Brisson 6k Ramirez, 2001;  Del Negro et al, 2002a; Abdala et al, 2004; Pena et al, 2004; Smith et al, 2004). At appropriate concentrations (EC the I  NAP  50  = 2 - 3 p.M), riluzole has been shown to be effective in selectively blocking  in brainstem transverse slice and isolated brainstem-spinal cord (en bloc) preparations  (Urbani 6k Belluzzi, 2000)(Del Negro et al, 2005). Flufenamic acid (FFA), a non-steroidal antiinflammatory drug, blocks the I  CAN  current  when applied at 100 - 500 pM in brainstem  transverse slice preparations (Gogelein et al, 1990; Pena et al, 2004; Del Negro et al, 2005)(Cho et al, 2003)(Pace et al, 2007a)(Pace et al, 2007b). Both drugs are commonly used in studying the contribution of I  NAP  and I  CAN  to respiratory rhythm generation.  1.2. Phytogeny and respiratory rhythm generation 1.2.1. Do phylogenetic differences exist in the roles of I  NAP  and ICAN in respiratory  rhythm generation? Hypothermic respiratory arrest and recovery from arrest originates at the respiratory CRG in the medulla (Mellen et al, 2002; Tattersall 6k Milsom, 2003). Therefore, the ability of adult hamsters and the inability of adult rats to restart breathing after hypothermic respiratory arrest may reflect a fundamental difference in the mechanism of respiratory rhythm generation in these 4  two species. Since I  NAP  and ICA facilitate inspiratory burst generation, they may be important in N  the recovery from hypothermic respiratory arrest by providing drive to the C R G to initiate breathing. Because adult hamsters are able to recover from respiratory arrest, we hypothesize that hamsters have a greater dependence on I  NaP  and I  CAN  for respiratory rhythm generation than rats.  1.3. Ontogeny and respiratory rhythm generation In rats, a developmental window exists where the ability to recover from hypothermic respiratory arrest is lost as the rat ages (Marshall, 2005). This loss of autoresuscitation ability implies that there are changes occurring in the CRG with development. We hypothesize that I  NaP  and ICAN are involved with initiating breathing after arrest and therefore asked if the reliance on I  NaP  and ICAN for rhythm generation changes during development. Several maturational changes  occur in the PBC in the first 12 days after birth: increased expression of excitatory neurotransmitters as well as a reversal of GABA receptor-mediated modulation (Wong-Riley 6k A  Liu, 2005)(Liu 6k Wong-Riley, 2004). The switch in the expression of GABA receptor subunits at A  12 PND results in a change in the GABA signal (via GABA ) from depolarizing to hyperpolarizing A  (Liu 6k Wong-Riley, 2004). [This GABAergic switch is distinct from the CI" current reversal which occurs at about embryonic day 19 due to increased CI" ion extrusion by the KCC2 cotransporter (Ren 6k Greer, 2006).] The membrane potential of respiratory neurons hyperpolarizes (to <-65 mV) over development so that I  NaP  is not active until the membrane is depolarized to -60 mV  (Richter 6k Spyer, 2001). The switch from GABAergic depolarization to hyperpolarization at 12 PND may contribute to the further hyperpolarizing of membrane potential of respiratory neurons in the PBC (Wong-Riley 6k Liu, 2005)(Liu 6k Wong-Riley, 2004). Inhibitory synaptic inputs act as a functional synaptic voltage clamp to hold the membrane of rhythmogenic neurons at voltages where I  NaP  and ICAN are not active (Richter 6k Spyer, 2001). It is unknown how these changes  might affect the generation of respiratory rhythm of the developing mammal.  1.3.1. Do ontogenetic differences exist in the roles of I  NlP  and I  CAN  in respiratory  rhythm generation? While most of the current literature examines the role of I  and ICAN i early post-natal n  NaP  development (Pena et al., 2004; Del Negro et al, 2005; Pace et al, 2007b; Pena 6k Aguileta, 2007), results from our laboratory suggest that the role of I  NaP  in rhythm generation may be changing as 5  rats mature (Marshall, 2005). The proportion of I  NAP  and IcAN-mediated bursting pacemaker  neurons in the PBC increase up to 15 PND (Pena et al, 2004; Del Negro et al, 2005); however, the increase in GABA -mediated inhibition in the PBC should counter the role of I A  NAP  and I  CAN  in  the expression of respiratory rhythm beyond 12 PND (Liu & Wong-Riley, 2004). Because rats lose the ability to autoresuscitate during development, a change may be occurring during development in the contribution of I  NAP  and ICAN to maintaining normal respiratory rhythm.  We hypothesize that during development changes are occurring in the C R G , particularly in the currents that engender intrinsic bursting (I  NAP  and I AN)> that cause the neonate to 'outC  grow' the ability to autoresuscitate. We hypothesize that the influence of l generation declines so that adult mammals depend less on I  NAP  and I  CAN  N a P  and I ^ N on rhythm  than neonatal mammals  for rhythm generation. To test our hypotheses that phylogenetic and ontogenetic differences occur in the roles of I  NAP  and ICANI  w  e  eliminated I  NAP  and ICAN in the arterially perfused in situ working heart-brainstem  preparation and examine the effect of the elimination of these currents on respiratory rhythm generation. Because both hamsters and young rats are capable of autoresuscitation, which may imply a greater dependence on I  NAP  and ICAN for rhythm generation, we expect that the elimination  of these currents will result in a greater decrease in respiratory output in mature hamsters and young rats than in mature rats.  6  Figure 1-1: Respiratory column location in the brainstem of mammals. Parasagittal (A) and transverse (B) views of brainstem adapted from Alheid et al. (2004) and Richter and Spyer (2001). Dashed line (A) delineates the pons from medulla. Abbreviations: 7, facial nucleus; 7n, facial nerve; A 5 , pontine noradrenergic group; BotC, Botzinger Complex; c V R G , caudal ventral respiratory group; IO, inferior olive; K F , Kolliker-Fuse nuclei; LPB, laternal parabrachial nucleus; LRt, lateral reticulum; M P B , medial parabrachial nucleus; M V e , medial vestibular nucleus; M o 5 , trigeminal motor nucleus; N A , nucleus ambiguus compactum; N T S , nucleus of the solitary tract; P B C , preBotzinger Complex; R O , nucleus raphe obscurus; R T Z / p F R G , region of the retrotrapezoid nucleus and parafacial respiratory group; r V R G , rostral ventral respiratory group; SCP, superior cerebellar peduncle; Sp5, spinal trigeminal nucleus; SpVe, spinal vestibular nucleus.  l _ K  t w  dominates  Figure 1-2: Bursting in respiratory neurons. (A) Simplified example of patch recording of the membrane potential (V ) of a bursting inspiratory cell and rectified, integrated motor output from hypoglossal nerve (XII). Example redrawn from Pace et al. (2007). (B) I -mediated pacemaker neuron bursting is represented in black. Grey line predicts action potential trajectory in the absence of I . Please see text for description. M  NaP  NaP  7  2. Methods Experimental protocols were approved by the Animal Care Committee of the University of British Columbia acting under the guidelines set by the Canadian Council for Animal Care (CCAC).  2.1. In situ working heart-brainstem preparation We used the in situ arterially perfused working heart-brainstem preparation (subsequently referred to as the in situ preparation) (Paton, 1996) as depicted in Figure 2-1. In brief, a SpragueDawley rat (Rattus nowegicus; U B C Rodent Breeding facilities) or hamster (Mesocricetus auratus; Charles River, Wilmington, MA) was anaesthetised deeply by halothane or isofluorane. The animal was transected caudal to the diaphragm and the anterior portion of the body was placed in chilled modified Ringer's solution (approximately 4 - 1 0 °C) bubbled with 95% Oj/5% C 0  2  carbogen gas. The dorsal braincase was removed and the animal was decerebrated at the precollicular level as indicated in Figure 2-2. The skin and any remaining abdominal viscera were removed and the diaphragm was removed carefully from the dorsal body wall and peeled back to expose the thoracic cavity. The descending aorta was separated from the dorsal body wall before the body wall was removed. The lungs were removed and the left phrenic nerve and pericardial cavity were cleared of any connective tissue. In a number of preparations, the right cervical vagus nerve (CVN) also was isolated by separating the nerve from the carotid artery in the neck and cutting the nerve at the most caudal point in the neck. The dissected preparation then was transferred to an acrylic recording chamber, placed dorsal side up, and secured at the head with ear bars. A double lumen catheter was inserted into the descending aorta to begin flow of perfusate to the preparation. Pump flow was calibrated prior to the experiments and flow rates for the groups are recorded in Table 2-1. The perfusate (modified Ringer's + 1.25% Ficoll® PM 70) was aerated with carbogen gas and warmed to 31 32°C by a heat exchanger then passed through bubble traps and a Millipore filter (25 uM) before entering the preparation. Upon warming of the preparation, the heart resumed beating, followed by inspiratory contractions of the diaphragm and intercostal muscles. The preparation was paralysed by adding a neuromuscular junction blocker, either rocuronium bromide (Zemuron, 2 mg) or vecuronium bromide (Norcuron, 0.6 mg), directly to the perfusate.  8  The left phrenic nerve was snipped at the diaphragm and both the cervical vagus and phrenic nerve (PHR) were connected to bipolar suction electrodes. The signals measured by the suction electrodes were passed through pre-amplifiers (FRAMP PRA-1,-2) then amplified (200 x) and filtered (500 Hz - 1kHz, F R A M P GPA-1). Perfusion pressure was measured by one lumen in the catheter connected to a physiological pressure transducer (Narco Scientific) connected to an amplifier (Gould Universal). Amplified nerve signals and perfusion pressure were sampled at 2.0 kHz and recorded by Windaq data acquisition software (DataQ Instruments, Akron, O H , USA).  2.2. Experimental protocol Only male rats and Syrian (also known as golden) hamsters were used. Rats lose autoresuscitation ability between 16 - 18 P N D (Marshall, 2005), so ages were chosen on either side of this developmental window. Rats were grouped as young rats (12 - 14 PND) or weaned rats (24 - 30 PND). The developmental stage of hamsters was matched to weaned rats (Clancy et al, 2001); weaned hamsters used were 23 - 40 P N D . Table 2-2 contains the mean weights of the different age groups. A l l age groups underwent the same protocol. The preparation was allowed to stabilize for 30 - 60 minutes before drugs were applied incrementally. Figure 2-3 shows a representative trace of C V N and P H R discharge activity measured at the beginning of an experiment using young rat and hamster preparations to validate the viability and stability of these preparations. Stable and viable preparations are characterised by an incrementing P H R discharge and observable threephase respiratory discharge from the C V N (Paton, 1996). As C V N discharge was used only for validation, it was not quantified. The drug regimen involved applying riluzole and F F A individually and in combination. When riluzole or F F A was applied individually, thirteen concentrations were administered, ranging 0.2 - 20 u M for riluzole and 0.25 - 25 u M for FFA. Because other authors have used larger concentrations of F F A in brainstem transverse slice preparations to completely eliminate ICAN  ( D ^ Negro et al, 2005; Pace et al, 2007a), a small number of experiments were run in which  concentrations of 25 - 100 u M FFA were applied only to weaned rat preparations in order to see if increasing F F A would result in a change in respiratory motor output. When riluzole and FFA were coapplied, only six concentrations were applied that had been calculated to result in a 5, 10, 9  15, 20, 25 or 50% decrease in frequency and amplitude from baseline values when the drug was applied individually (without correcting for vehicle controls). In all cases, sufficient time after each drug application was given in order for the response to stabilize (approximately 5 - 1 5 minutes depending on flow rate). Del Negro and colleagues (2005) showed that riluzole and F F A were effective in abolishing respiratory rhythm in in vitro transverse brainstem slice preparations. Application of substance P (excitatory neurotransmitter) rescued respiratory rhythm by increasing drive in the respiratory network. In order to increase respiratory drive in the in situ preparation, we increased the proportion of C 0  2  bubbling in the perfusate to 7% (93% 0 ) . In these experiments, the 2  preparation was initially perfused and allowed to stabilize with 5% C 0 perfusate for 30 - 60 2  minutes. The gas mixture was then switched to 93% 0 ^ 7 % C 0  2  and the preparation was  allowed to stabilize for another 20 - 30 minutes until it had reached a steady state. When the preparation had stabilized, I followed the same combined drug regimen (riluzole and FFA) as described for the preparations using 5% C 0 . 2  Riluzole (Sigma, St. Louis, M O ; 25 mg) was solubilized with 2 ml of 1.0 M hydrochloric acid and heated while stirring until all the riluzole dissolved. The solution was diluted to a concentration of 2 m M by adding 50 ml de-ionized distilled water (ddH 0). F F A (Sigma, St. 2  Louis, M O ) was solubilized with 100 m M N a O H and then titrated to p H 7.4 - 8.0 with 1.0 M and 0.1 M HCI. The solution was diluted to 5 m M by adding d d H 0 . For all experiments, vehicle 2  controls, using just the solvent for each drug, were run by adding the same volume of solution that had been added in the drug runs to correct for effects of increase perfusate volume and time.  2.3. Data and statistical analysis The data collected by Windaq was imported and analyzed in Spike2 software (v 4.24, Cambridge Electronic Designs, Cambridge, U K ) . Figure 2-4 shows how the variables measured were derived from the phrenic neurogram (PHR). Inspiratory duration, T was measured as the h  time between the onset and end of phrenic bursting; expiratory duration, T , was measured as the E  time between the end of a phrenic burst and the onset the following burst. The inverse of the sum of T[ and T was multiplied by 60 to calculate burst frequency (bursts min' )- Amplitude of the 1  E  phrenic burst was also measured as the peak of the burst in the rectified integrated neurogram. 10  Amplitude and frequency then were multiplied for a measure of "neural ventilation" (nV ). E  These variables were measured for the 60 seconds of steady-state activity preceding each drug application. The values for 60 seconds were averaged and normalized to the baseline value recorded before drug applications began; therefore, changes in fictive respiration in response to increasing concentrations of riluzole or FFA are expressed as a proportion of the baseline value (baseline=1.0). These normalised values were corrected for time by dividing the experimental values by the vehicle control values. SigmaStat was used for statistical analysis. The data were not distributed normally and an arcsine square root transformation was not possible due to the proportion values being >1.0; therefore, the data were ranked and then tested using a two-way repeated measures A N O V A with a Student-Newman-Keuls post-hoc test with P < 0.050 considered significant. For the experiments using high concentration of FFA on only weaned rat preparations, the data were ranked and tested using a one-way repeated measured A N O V A and Student-Newman-Keuls post-hoc test (P < 0.050).  11  Table 2-1: The range, mean and median perfusate flow rate values to in situ preparation. Age group Young rats Weaned rats Weaned hamsters  Range (ml/min) 8-18 30-40 19-38  Mean ± SD (ml/ min) 12.9 ± 2 . 7 34.4 ± 4.3 29.0 ± 5.3  Median (ml/min) 12 32 30  Table 2-2: The range, mean and median weight values of young rats, weaned rats and weaned hamsters used in the in situ preparation. Age group Young rats Weaned rats Weaned hamsters  Range (g) 20-41 68 - 107 33 - 100  Mean ± SD (g) 28.1 ± 4 . 0 85.7 ± 15.7 62.9 ± 18.9  Median (g) 27.5 82 62  12  7  Figure 2-1: Schematic diagram of the in situ working heart-brainstem preparation. The transected, decerebrated rodent is retrogradely perfused with a modified Ringer's solution containing 1.25% Ficoll that has been aerated with carbogen gas, warmed to 31 - 32 °C and filtered. Respiratory motor output is measured by recording phrenic nerve activity and cervical vagus nerve activity with suction electrode. These signals are further amplified, filtered and recorded. Schematic diagram is redrawn and adapted from Potts et al. (Potts et al, 2000).  Figure 2-2: Approximate location of decerebration in a sagittal section of the rat brain. The decerebration removes structures of the brain anterior to the thalamus and retains the areas of the thalamus, colliculi, pons, cerebellum, brainstem and spinal cord. Abbreviations: 3V, third ventricle; 4V, fourth ventricle; C B , cerebellum; C C , corpus callosum; Cx, cerebral cortex; IC, inferior colliculus; T h , thalamus; Pons, pons; SC, superior colliculus. Adapted from Paxinos and Watson (Paxinos & Watson, 1986).  13  .PHR  5 sec  B  JCVN  J  5 sec  JCVN CVN  A  JPHR PHR  I I i < |  | |  i  <| 5 sec  Figure 2-3: Representative neurograms of respiratory activity in young rat and hamster in situ preparations. (A) Expanded view of (B) young rat and (C) hamster raw and rectified, integrated (j) cranial vagus nerve (CVN) and phrenic nerve (PHR) neurograms measured to deduce the stability of the in situ preparation. Viable in situ preparations are characterised by C V N activity during the preinspiratory (Pre-I) period preceding the PHR burst, during the inspiratory (Insp) phase during the PHR burst, during the post-inspiratory (Post-I/El) phase following the PHR burst but quiescence during the period between the Post-I and Pre-I phases, known as the expiratory phase (E2) (Paton, 1996). 14  Figure 2-4: Variables measured from a phrenic neurogram. The top trace is the rectified integrated trace of phrenic nerve activity and the bottom trace is the raw neurogram. Abbreviations: T inspiratory duration; T , expiratory duration. h  E  15  3. Results In this study, riluzole (I  NAP  blocker) and flufenamic acid (FFA; l  CAN  blocker) were applied to  the in situ preparation in young (12 - 14 PND) and weaned (23 - 30 PND) rats and weaned hamsters (>23 PND) to test the phylogenetic and ontogenetic differences in the contribution of I  NAP  and ICAN to respiratory rhythm generation. The ages of the rat groups were chosen based on a  hypothesized critical developmental period between 16 - 18 PND in rats when the ability to spontaneously recover from hypothermia-induced respiratory arrest is lost (described in section 1.1). Hamster ages were chosen to match the developmental stage of the weaned rat group. Based on our hypotheses, we expected that respiratory output of the groups capable of autoresuscitation from hypothermic respiratory arrest, young rats and weaned hamsters, would decrease to a greater extent than the motor output from weaned rat preparations when I  NaP  and ICAN were eliminated.  The mean and range of starting frequency of bursts measured from the phrenic nerve for young rat, weaned rat and hamster preparations are recorded in Table 3-1. Hamster preparations tended to have much faster bursting rates than either groups of rats, but hamster preparations also had more variation in starting bursting rate.  3.1. Riluzole application Riluzole blocks specifically I p when applied at low concentrations. At the beginning of Na  this study, riluzole had not been applied to the in situ preparation. In brainstem transverse slice preparations, concentrations of 1 - 200 U.M riluzole had been applied (Del Negro et al, 2002a; Pena et al, 2004; Del Negro et al, 2005). We began applying riluzole at 0.2 pM to 20 pM which yielded a reduction in motor output; therefore, higher concentrations were not applied. Not all preparations continued generating respiratory motor output for all concentration of riluzole; Table 3-2 shows the number of preparations still producing phrenic motor output at the indicated concentration of riluzole. As the concentration of riluzole increased to 20 pM, only 27% of young rats continued to generate motor output. All weaned rat preparations continued generating phrenic bursts to 20 pM; however, no hamster preparation continued respiratory rhythm generation past 14 pM. Figure 3-1 shows examples of continuous tracings from the raw and rectified integrated neurogram of phrenic nerve discharge in young and weaned rats and 16  weaned hamster at representative concentrations. Neither the young rat nor the hamster showed continued bursting at 20 uM, but both showed profound decreases in frequency and amplitude at 8 uM where only an amplitude decrease was observed in the weaned rat. Burst frequency, amplitude, and neural ventilation (nV ) were quantified, corrected for E  vehicle controls and presented as relative to baseline (= 1.0) in Figure 3-2. Frequency (Figure 3-2A) did not change significantly from baseline values (0 uM) at low concentrations for either age of rat. However, as concentration increased, frequency decreased significantly from baseline in the young rats but not in the weaned rats leading to a significant difference between the ages by 10 uM. In contrast, the frequency of phrenic bursts profoundly decreased at very low concentrations in hamster preparations. For instance, 50% of baseline frequency was reached by 1 - 2 uM riluzole in hamsters; in young rats, 1 0 - 1 2 uM riluzole was required for this degree of change. Weaned rat frequency never reached 50%. A statistically significant difference existed between hamsters and both age groups of rats at low concentrations. Amplitude (Figure 3-2B) did not change significantly in young or weaned rat preparations at low concentrations. However, as concentration increased, the amplitude in young rat preparations declined significantly and became significantly different from the amplitude in weaned rat preparations. The amplitude of phrenic bursts decreased significantly in hamster preparations after 6 uM. Between 6 and 10 uM, hamster preparations were significantly different from both ages of rats. Neural ventilation, nV , (Figure 3-2C) in weaned rats did not change significantly from E  baseline until 18 uM. Young rats significantly decreased nV at 8 uM, however no significant E  nIn-  difference existed between the ages until 12 uM. Hamster preparations decreased significantly from baseline at low concentrations.  To depict the changes in T[ and T , pyramid plots are shown in Figure 3-3. In a pyramid E  plot, the upward slope from amplitude = 0 to the peak represents inspiratory duration (T[). The downward slope from peak amplitude to amplitude = 0 represents expiratory duration (T ). To E  calculate the values for T[ and T , normalized T[ and T means corrected for vehicle controls (in E  E  Appendix Table 6-1) were multiplied by the group mean absolute baseline value. In both ages of 17  rats, T[ did not deviate significantly from baseline values (Figure 3-3A, B). Conversely in young rats, T steadily increased with increasing concentrations of riluzole (downward slope shifts right E  along x-axis). In weaned rats, T also increased but changed very little. In response to increasing E  concentrations of riluzole, rat preparations preferentially decreased frequency by elongating the interburst interval. Hamsters (Figure 3-3C) increased both T, and T (peak and downward slope E  shift right along x-axis) which resulted in the observed change in frequency.  3.2.Flufenamic acid application Similar to riluzole, F F A had not been applied to the in situ preparation; previously reported concentrations applied to brainstem transverse slice preparations ranged 100 - 500 u M FFA (Pena et al, 2004; Del Negro et al, 2005). We applied F F A starting at 0.25 U.M and increased the concentration of F F A until a significant response was obtained in the raw data. Most preparations of weaned rats and hamsters continued producing fictive respiratory motor output until the highest concentration of FFA (25 uM) (Table 3-3). Furthermore, sixty percent of the young rat preparations also continued to generate phrenic motor output at 25 u M FFA. Representative raw and rectified, integrated neurograms are presented in Figure 3-4. Unlike the riluzole trials, all groups continue bursting at the highest concentration and all groups showed frequency increasing with increasing concentrations of FFA. This increase in frequency also was observed during vehicle control runs. Similarly, amplitude in all three groups decreased over time, which was also observed during vehicle controls. Figure 3-5 shows the changes in normalized frequency (A), amplitude (B), and nV (C), E  corrected for vehicle controls, in response to increasing concentrations of FFA. Burst frequency (Figure 3-5A) was not significantly affected by the addition of F F A in any of the groups. Amplitude (Figure 3-5B) decreased with increasing F F A concentration in all groups. Weaned rats decreased amplitude significantly from baseline at very low concentrations; however, amplitude was maintained at about 60% of baseline until 25 u M . Amplitude slowly decreased in young rat preparation and was significantly different from baseline after 10 u M . A significant difference exists between young and weaned rats at low concentrations of F F A . Hamster preparations maintained phrenic burst amplitude near baseline values. 18  Neural ventilation decreased significantly in weaned and young rats, particularly at higher concentrations, but nV was maintained around baseline in hamsters (Figure 3-5C). E  Pyramid plots in Figure 3-6 depict the changes in Tj and T in response to increasing E  concentrations of FFA and were calculated as described in section 3.1 with the data in Appendix Table 6-2. In young rat preparations (Figure 3-6A), T, decreased to 60% of baseline and T  E  decreased to 35% of baseline. In contrast, T in weaned rat preparations (Figure 3-6B) did not {  change significantly from baseline; however, T  E  tended to increase as FFA concentration  increased. In hamsters (Figure 3-6C), T] and T remained the same with increasing concentrations E  of FFA. Greater concentrations of FFA have been applied in brainstem transverse slice preparations to completely eliminate IQ^ (Del Negro et al, 2005; Pace et al, 2007a); therefore, a small number of experiments were run in which concentrations of 25 - 100 pM FFA were applied only to weaned rat preparations in order to see if increasing FFA would result in a change in respiratory motor output. When FFA was applied to weaned rat preparations up to a concentration of 100 pM (Figure 3-7), only one third of preparations continued generating motor output while two thirds of the preparations ceased discharge at 50 pM. Frequency and amplitude significantly declined with increasing concentration; therefore, their product, nV , E  also  significantly declined.  3.3. Riluzole and FFA coapplication We applied riluzole and FFA in combination to test the response of rats and hamsters to the elimination of both I _ and N  P  IQAN-  We expected that young rats and hamsters would have a  greater decrease in Active breathing resulting from a hypothesized greater dependence on I  NaP  and  ICAN f ° respiratory rhythm generation. Six concentrations of the riluzole and FFA combination r  were applied, which had been calculated from the experiments where the drugs were applied individually to result in a 5, 10, 15, 20, 25 or 50% decrease in frequency and amplitude from baseline values.  19  3.3.1. Coapplication at 5% C 0  2  Adding riluzole and FFA in combination to rat and hamster in situ preparations was effective in abolishing respiratory rhythm in some preparations and Table 3-4 gives the number of preparations that continued to produce phrenic bursting at the indicated concentrations of riluzole and FFA. No young rat preparations continued generating motor output at 20 uM riluzole + 15 uM FFA. Only 30% of weaned rats and 50% of hamster preparations continued generating bursts after the final concentration. In the example neurograms shown in Figure 3-8, neither the young rat or hamster preparation continued breathing after the coapplication of 20 uM riluzole + 15 uM FFA. The weaned rat preparation continued to breathe and frequency tended to remain at baseline values; however, amplitude was profoundly decreased. Before respiratory rhythm was abolished in young rat and hamster preparations, frequency of phrenic bursts was profoundly decreased and amplitude of phrenic bursts decreased. Normalized frequency, amplitude, and nV , corrected for vehicle controls, of the phrenic E  motor output are presented in Figure 3-9. Similar to riluzole trials, burst frequency (Figure 3-9A) of hamsters decreased significantly at lower concentrations of riluzole and FFA; 50% of baseline values was obtained at 7.5 uM riluzole + 5 uM FFA. The frequency of phrenic bursts also decreased significantly in young rat preparations. Weaned rat preparations reduced frequency to below 50% at 20 uM riluzole +15 uM FFA. Significant differences exist between young and weaned rats at higher concentrations as weaned rats maintained frequency at concentrations where young rat burst frequency declined. The amplitude (Figure 3-9B) of phrenic bursting decreased significantly in all groups. Young and weaned rat preparations decreased amplitude at low concentrations, particularly in young rats (to 14% of baseline). In rats, the phrenic burst amplitude typically reached zero while frequency decreased less. Hamster preparations decreased amplitude the least. Neural ventilation (Figure 3-9C) significantly decreased in all groups in response to increasing concentrations of riluzole and FFA. The greatest decrease in nV occurred in weaned E  rat preparations where nV reached <10% of baseline. However, the drop in nV was due to E  E  different components (though the effects are offset so nV was similarly affected in all groups). In E  20  hamster preparations, decreased frequency contributed more to the decrease in nV ; in rat E  preparations, decreased amplitude contributed to the decrease in nV . E  The response of n V in response to individual application of riluzole and FFA were added E  to calculate expected nV values based on the assumption that riluzole and FFA effects are E  additive. Figure 3-10 compares the expected values with the actual response of nV . In all groups, E  the actual response to the coapplication of riluzole and FFA was more profound than expected. The only exception was in hamster preparations at 6 pM RIL + 2.5 pM FFA where nV was E  expected to be lower than the actual result. Pyramid plots (Figure 3-11) for young and weaned rats and weaned hamster preparations were calculated using data from Appendix Table 6-3 as described in section 3.1. In young and weaned rat preparations (Figure 3-llA,B), T[ stayed at baseline values. In both young and weaned rat groups, T increased as concentrations of riluzole and FFA increased. In hamster preparations E  (Figure 3-11C), T] remained around baseline values and T increased dramatically up to 37x E  baseline values at 10 pM riluzole + 8 pM FFA, after which T values decreased. In response to E  increases concentrations of riluzole and FFA in combination, rats and hamsters decreased frequency by increasing the interburst interval (T ). E  3.3.2. Coapplication at 7% C 0  2  Del Negro and colleagues (2005) showed that increasing drive to the respiratory network (by application of substance P) after the abolition of respiratory rhythm by coapplication of riluzole and FFA rescued respiratory rhythm. In order to increase respiratory drive in the in situ preparation, we increased the proportion of CO bubbling in the perfusate to 7% (93% 0 ) and z  2  expected that respiratory rhythm would be greater during coapplication of riluzole and FFA than the preparations exposed to 5% CO . Preparations were exposed to perfusate equilibrated with z  7% C 0 (93% 0 ) in order to increase the respiratory drive when the combination of riluzole and 2  2  FFA were administered. For these preparations, 15% of young rat, 63% of hamster and all weaned rat preparations continued generating motor output after the final concentration of the drug combination (A Amplitude is the relative change in phrenic burst amplitude. Dashed line represents baseline Tl. Please note different times axis for hamsters. 21  Table 3-5). At 5% C O , no young rat preparations continued bursting after the final z  concentration of the cocktail (Table 3-4). The baseline values for preparations perfused with 7% C 0 were calculated by dividing 2  the 7% C 0 (high drive) value by the 5% C 0 (normal drive) value prior to drug application. 2  2  Table 3-6 shows the frequency, T, and T baseline (absolute) values for preparations at 5% or 7% E  C 0 . Frequency in rat preparations tended to increase when exposed to 7% C 0 (significantly in 2  2  young rats preparations) while frequency in hamsters appeared to stay the same. These changes in frequency appear to be due to both shortening of the inspiratory and expiratory duration. Frequency (Figure 3-12) of phrenic bursts in all high drive preparations remained higher than the frequency in normal drive preparations. Within all groups, phrenic burst frequency decreased at a similar rate for both 5% and 7% C 0 following coapplication of riluzole and FFA. 2  In young rats (Figure 3-12A), frequency was significantly higher at low concentrations for 7% C 0  2  than 5% C 0 . This difference disappeared at higher concentrations. No significant difference 2  exists between high drive and normal drive preparations in weaned rats (Figure 3-12B) and hamsters (Figure 3-12C). Increasing respiratory drive by stimulating chemoreceptors did not negate the effect of riluzole or FFA in weaned rats or hamsters on frequency. The amplitude of phrenic nerve bursts (Figure 3-13) in young rat preparations (Figure 3-13A) decreased at high and normal drive at a similar rate. In weaned rats (Figure 3-13B), the amplitude of high drive preparations was significantly greater than the amplitude values for normal drive preparations. The weaned rat preparations at 7% C O maintained amplitude z  around baseline values, while the normal drive amplitude fell from baseline. In hamsters (Figure 3-13C), phrenic burst amplitude of high and normal drive preparations was the same and remained around baseline values. No significant difference existed between 5% C 0 and 7% C 0 2  2  preparations for young rats or hamsters. In all groups, nV (Figure 3-14) decreased significantly from baseline values in high drive E  preparations as concentrations of the drug combination increased. In weaned rat preparations (Figure 3-14B), ventilation was increased significantly in 7% C 0 . In young rats and hamsters 2  (Figure 3-14A, C), the ventilation of high drive preparations was very similar to the values for 22  normal drive preparations and decreased from baseline values at the same rate. Exposing in situ preparations to 7% C O and coapplication of riluzole and F F A significantly increased ventilation z  in weaned rats over the majority of concentrations and seemed to have no significant effect on the response of young rats or hamsters to riluzole and FFA.  23  Table 3-1: Mean and the range of baseline phrenic nerve burst frequency for young rats, weaned rats and weaned hamsters. The values are ± standard error. Age group Young rat Weaned rat Weaned hamster  Mean (bursts/min) 19.4 ± 1.0 18.1+0.9 46.6 ± 4.2  Range (bursts/min) 6.4-51.5 6.6 - 35.3 13.4 - 128.6  Table 3-2: The number of animals with phrenic nerve activity at indicated concentrations of riluzole.  [R1L] (uM) 0.0 0.2 1.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0  Nu mber of animals Young Weaned Weaned hamsters rats rats 11 6 6 11 6 6 11 6 6. 11 6 6 11 6 6 11 5 6 11 3 6 2 10 6 1 6 5 1 3 6 0 6 3 0 3 6 6 0 3  24  Figure 3-1: Thirty second representative raw (PHR) and rectified integrated (JTHR) neurograms of P H R nerve activity in response to increasing concentrations of riluzole. Top series is from a young rat (12 PND), middle series from a weaned rat (25 PND), bottom series from a weaned hamster (26 PND).  25  0  5  10  15  20  25  [ R i l u z o l e ] (uM)  Figure 3-2: The change in phrenic nerve burst frequency (A), amplitude (B), and neural ventilation (nV , E  C), corrected for vehicle controls, in response to increasing concentrations of  riluzole. Young rats (n=ll) are represented by circles (•); weaned rats (n=6) are represented by squares (•); weaned hamsters (n=6) are represented by diamonds (•). Open shapes indicate significant differences from 0 pM (denoted by grey dashed line). Asterisks (*) denote significant differences between young and weaned rats. Daggers (t) denote significant difference between young rats and weaned hamsters; double daggers ($) denote significant differences between weaned rats and hamsters. 26  Young rats  0 u M RIL 0.2 u M RIL  1 u M RIL 2 u M RIL 4 u M RIL 6 uM RIL 8 u M RIL  10 u M RIL 12 u M RIL 14 u M RIL 16 u M RIL 18 u M RIL 2 0 u M RIL  2  3  4  Time (sec)  Figure 3-3: Pyramid plots for young rats (A), weaned rats (B) and weaned hamsters (C) for increasing concentrations of riluzole (RIL). A Amplitude is the relative change in phrenic burst amplitude. Dashed line represents baseline TV Please note different times axis for hamsters. 27  Table 3-3: The number of animals wiith phrenic nerve activity at indicated concentrations of FFA.  Concentration (uM) 0.0 0.25 1.25 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0  Number of animals Young rats Weaned Weaned rats hamsters 10 7 7 10 7 7 10 7 7 10 7 7 10 7 7 10 7 7 9 7 7 8 7 • 7 8 7 7 6 7 7 6 7 7 6 6 7 6 6 7  28  Figure 3-4: Thirty second representative raw (PHR) and rectified integrated (JTHR) neurograms of P H R nerve activity in response to increasing concentrations of FFA. Top series is from a young rat (14 PND), middle series from a weaned rat (25 PND), bottom series from a weaned hamster (29 PND).  29  Figure 3-5: The change in phrenic nerve burst frequency (A), amplitude (B), and neural ventilation (nV , E  C), corrected for vehicle controls, in response to increasing concentrations of  FFA. Young rats (n=10) are represented by circles (•); weaned rats (n=7) are represented by squares (•); weaned hamsters (n=7) are represented by diamonds (•). Open shapes indicate significant differences from 0 uM (denoted by grey dashed line). Asterisks (*) denote significant differences between young and weaned rats. Daggers (t) denote significant difference between young rats and weaned hamsters; double daggers (t-) denote significant differences between weaned rats and hamsters. 30  Young rats  0 uM FFA 0.25 uM FFA 1.25 uM FFA 2.5 \M FFA 5.0 FFA 7.5 FFA 10 uM FFA 12.5 uM FFA 15 uM FFA 17.5 uM FFA 20 uM FFA 22.5 uM FFA 25 fiM FFA 1  2  3  4  5  T i m e (sec)  Weaned hamsters  0.5  1.0  2.0  T i m e (sec)  Figure 3-6: Pyramid plots for young rats (A), weaned rats (B) and weaned hamsters (C) for increasing concentrations of FFA. A Amplitude is the relative change in phrenic burst amplitude. Dashed line represents baseline T_. Please note different times axis for hamsters. 31  0.0  J  1  1  1  1  r—  0  25  50  75  100  [FFA] u M  Figure 3-7: Frequency, amplitude and neural ventilation {nV ) all declined as F F A concentration E  increased in weaned rat preparations (n=6). Open shapes indicate significant difference from 0 u M . Table 3-4: Number of preparations producing respiratory rhythm during coapplication of riluzole and FFA.  Concentration [RILMFFA] 0 uM+0 u M 6 uM+2.5 u M 7.5 uM+5 u M 9 uM+6 u M 10 uM+8 p M 12pM+10pM 20pM+15pM  Number of animals Weaned Young Weaned hamsters rats rats 10 8 16 8 16 9 9 7 14 9 13 7 6 9 7 6 10* 4 0 3 4  * the values from one preparation in the weaned rat group only had 0 u M RIL + FFA, 12 uM+10 u M and 20 uM+15 u M applied. 32  7.5uM RIL/5uM F F A  OuM  I  JPHR  10uM RIL/8u.M F F A  20|iM RIL/15uM F F A  u  PHR 5 sec  |!PHR  J U U U U U L  PHR r|i^!;Li.iLi^Lt-iLli-il<,  jUXLAXlUUl  ^ K J u U U J U j ^  •|.p||iH^..|iii|i»l j|.i'i|i>i^i j>  ^ ^ i ^ ^ ^ . j ,  l  l  ^ j ^ ^ ^ ^ j ^ h . >..#.•  )i  H  5 sec  JPHR Jl  •y+t# -*,>+Y'  V-V""  1  PHR 5 sec  Figure 3-8: Thirty second representative raw (PHR) and rectified integrated (JPHR) neurograms of PHR nerve activity in response to increasing concentrations of riluzole and FFA. Top series is from a young rat (12 PND), middle series from a weaned rat (27 PND), bottom series from a weaned hamster (25 PND).  33  Figure 3-9: The change in phrenic nerve burst frequency (A) and amplitude (B), and neural ventilation (nV , C ) corrected for vehicle controls, in response to increasing concentrations of riluzole and FFA. E  Young rats (n=16) are represented by black bars (•); weaned rats (n=10) are represented by light grey bars (H); weaned hamsters (n=8) are represented by dark grey bars (•). Hashed bars indicate significant differences from 0 u M (denoted by grey dashed line). Asterisks (*) denote significant differences between young and weaned rats. Daggers (f) denote significant difference between young rats and weaned hamsters; double daggers (t-) denote significant differences between weaned rats and hamsters. 34  [FFAJ(jiM)  2.5  5  6  8  10  15  2.5  5  6  8  10  15  2.5  5  6  8  10  15  Figure 3-10: Expected values for neural ventilation (nV ) based on additive action of riluzole and E  FFA compared to the actual nV for young rats (A), weaned rats (B) and weaned hamsters (C). E  Dashed line = baseline =1.0.  35  Young rats  0 nM/O u M 6 u M RIL/2.5 u M F F A 7.5 u M R I L / 5 u M F F A 9 u M RIL/6 u M F F A 10 u M RIL/8 M M F F A 12 u M R I L M O u M F F A 20 u M R I L / 1 5 n M F F A  6  8  10  12  14  16  18  Time (sec)  Figure 3-11: Pyramid plots for young rats (A), weaned rats (B) and weaned hamsters (C) for increasing concentrations of riluzole and FFA combination. A Amplitude is the relative change in phrenic burst amplitude. Dashed line represents baseline T|. Please note different times axis for hamsters. 36  Table 3-5: Number of preparations exposed to 7% C 0 producing respiratory rhythm during coapplication of riluzole and FFA. 2  Concentration [RILMFFA] 0 pM+0 pM 6 uM+2.5 uM 7.5 uM+5 pM 9 pM+6 pM 10 uM+8 pM 12 pM+10 pM 20uM+15 pM  Number of animals Weaned Young Weaned hamsters rats rats 6 8 13 6 8 13 8 12 6 10 6 8 6 8 7 6 8 7 2 5 6  Table 3-6: Alterations in phrenic nerve burst frequency, T and T of young rat, weaned rat and weaned hamster preparations exposed to 5% C O or 7% C 0 . Values are expressed as means±standard error. N=number of preparations. (  z  Young rat N-13 Weaned rat N=6 Hamster N=8  Frequency (bursts/min) 5% 7% 17.5 ± 1.78 22.6 ± 2.22  T, (sec) 5% 0.48 ± 0.02  E  2  7% 0.41 ± 0.04  T (sec) 5% 3.67 ±0.67  7% 2.80 ±0.53  E  17.1 ±6.4  21.4 ±3.4  0.82 ± 0.33  0.64 ±0.21  3.41 ±2.58  2.23 ± 0.65  29.4 ± 4.6  29.2 ±3.1  0.57 ± 0.07  0.60 ± 0.08  1.79 ±0.27  1.66 ±0.28  37  Figure 3-12: Changes in frequency, corrected for vehicle controls, for young rat (A), weaned rat (B) and hamster (C) preparations exposed to perfusate equilibrated with 5% C 0 or 7% C 0 and coapplication of riluzole and FFA. Preparations at 5% are represented in black. Preparations at 7% for young rats (n=13), weaned rats (n=6) and hamsters (n=8) are represented in grey. Hashed bars indicate significant difference from value at 0 pM (denoted by grey dashed line). Asterisks (*) denote significant differences between 5% and 7% C 0 preparations. 2  2  2  Figure 3-13: Changes in amplitude, corrected for vehicle controls, for young rat (A), weaned rat (B) and hamster (C) preparations exposed to perfusate equilibrated with 5% C O or 7% C 0 and coapplication of riluzole and FFA. Preparations at 5% are represented in black. Preparations at 7% for young rats (n=13), weaned rats (n=6) and hamsters (n=8) are represented in grey. Hashed bars indicate significant difference from value at 0 pM (denoted by grey dashed line). Asterisks (*) denote significant differences between 5% and 7% C 0 preparations. z  2  2  38  Figure 3-14: Changes in neural ventilation (nV ), corrected for vehicle controls, for young rat (A), weaned rat (B) and hamster (C) preparations exposed to perfusate equilibrated with 5% C 0 or 7% C 0 and coapplication of riluzole and FFA. Preparations at 5% are represented in black. Preparations at 7% for young rats (n=13), weaned rats (n=6) and hamsters (n=8) are represented in grey. Hashed bars indicate significant difference from value at 0 uM (denoted by grey dashed line). Asterisks (*) denote significant differences between 5% and 7% C 0 preparations. E  2  2  2  39  4. Discussion We hypothesize that phylogenetic and ontogenetic differences occur in the roles of I and  ICAN  such that hamsters and young rats have a greater reliance on I  rhythm generation than weaned rats. Riluzole (an I  NaP  NaP  and  ICAN  f ° respiratory  blocker) and FFA (an  ICAN  blocker) were  NaP  r  applied to the in situ preparation in young (12 - 14 PND) and weaned (23 - 30 PND) rats and weaned hamsters (>23 PND). Our results indicate that hamsters depend on I  NaP  and  ICAN  t o  a  greater extent for  respiratory rhythm generation than rats. The fictive breathing frequency of hamster preparations significantly decreased during application of riluzole and coapplicaton of riluzole and FFA to the in situ preparation. This change in frequency was significantly different from the response of young and weaned rats. Fictive respiration amplitude in hamsters was less affected by the coapplication of riluzole and FFA than in rats. Total neural ventilation was significantly lower in hamsters than rats during application of riluzole, but no significant difference exists during the coapplication of riluzole and FFA. The frequency, amplitude and neural ventilation of young rats also tended to be affected more by the application of riluzole and FFA than those of weaned rats. For instance, young rats, like hamsters, depend to a greater extent on I  NaP  and I  CAN  for respiratory rhythm generation than  weaned rats. These data indicate that a change in the role of I  NaP  and I  CAN  in the C R G occurs  during development. Increasing excitatory drive to the C R G by increasing C 0  2  significantly increased  ventilation in weaned rats only. These data suggest that in weaned rats other sources of respiratory drive may compliment the roles of I  NaP  and  ICAN-  4.1. Technical considerations Riluzole binds to Na and K channels in their inactivated state (but not their resting or +  +  open state) (Benoit 6k Escande, 1993)(Benoit 6k Escande, 1991)(Doble, 1996). Although riluzole inhibits both Na and K currents, it preferentially binds to Na channels and is estimated to have +  +  +  300 times more affinity for inactivated state Na channels than resting channels (Benoit 6k +  Escande, 1991)(Benoit 6k Escande, 1993). By preferentially binding the inactivated state of Na  +  40  channels, riluzole shifts the membrane potential to a more hyperpolarized voltage, resulting in the inactivation of I . [As described in section 1.3, I NaP  NaP  is active at more depolarized potentials.] (Ptak  et al, 2005; Feldman & Del Negro, 2006). Riluzole also interferes with glutamate signalling, which further affects the activation of Ca  2+  and Na currents (Doble, 1996). However, ample +  evidence shows that at low concentrations for short periods of time, riluzole is specific for I  NaP  (EC50 = 3 pM for in vitro brainstem transverse slices, = 2 pM for cortical slices) (Urbani 6k Belluzzi, 2000)(Del Negro et al, 2005). At the concentrations used in this study (0.5 pM - 20 pM), while riluzole may have been acting on several channels and currents, it will have certainly blocked I . NaP  The channels that produce the ICAN current are most likely members of the TRPM family (transient receptor potential-melastatin) which are inhibited by FFA (100 pM) (Launay et al, 2002; Guinamard et al, 2004). In in vitro transverse slices, bath applying 100 pM FFA was not sufficient to completely eliminate 1 ^ (Pace et al, 2007a). Concentrations of FFA that do completely abolish 1 ^ also will block gap junctions reversibly (EC = 40 pM - 47 pM) (Srinivas 50  6k Spray, 2003)(Harks et al, 2001), reversibly inhibit Ca -dependent and voltage-dependent CI" 2+  channels (White 6k Aylwin, 1990), and stimulate or inhibit (depending on concentration) large conductance Ca -activated K channels which may influence neuronal firing (Ottolia 6k Toro, 2+  +  1994)(Li et al, 1998)(Farrugia et al, 1993)(Kochetkov et al, 2000). However, many investigators use FFA with the understanding that results regarding the elimination of I  CAN  must be cautiously  interpreted due to the multiple and incomplete actions of FFA. Previous studies have reported that riluzole only affected the respiratory frequency of hypoxia-induced gasping (Paton et al, 2006). The frequency response to riluzole seen in our hamster and young rat preparations might suggest therefore that our preparations are hypoxic and the rhythm we recorded was gasping, and not normoxic breathing. For this reason, we recorded from the C V N to validate the stability and viability of the hamster and young rat in situ preparations (see Methods for explanation). The recordings of C V N activity (Figure 2-3B, C) showed that our young rat and hamster preparations were stable and viable because the preparations exhibited the three neural phases of respiration (inspiratory, post-inspiratory and expiratory). Therefore, we can confirm that the rhythms we recorded were not gasping and 41  attribute any decreases in frequency to the action of riluzole on I  NaP  (and other currents) rather  than poor stability of the preparations. In our measurements of respiratory variables, frequency of phrenic discharge indicates the rhythm outputted by the CRG. Therefore, to change frequency, thus rhythm, requires bringing membrane potential to threshold either more quickly or more slowly. Amplitude, in this study, does not refer to rhythm but to the net motor output generated by the entire respiratory network. To change amplitude implies that either fewer respiratory neurons are active or fewer repetitive bursts occur per breath per bursting neuron. Therefore, if elimination of I  NaP  and I  CAN  results in a  change in frequency, rhythmogenesis at the CRG would be affected; whereas, a decrease in amplitude would indicate that the elimination of I  NaP  and IQ^N has decreased the excitability of  respiratory network.  4.2. Phylogenetic differences in I  NaP  and ICAN contribution to rhythm generation  Hamsters are permissive hibernators that require specific cues, such as water deprivation and shorter photoperiods, to induce hibernation (Ueda 6k Ibuka, 1995) while rats do not show this type of dormancy. Hamsters are also fossorial species exhibiting adaptations to living in hypoxic, hypercapnic burrow environments (Walker et al, 1985) in addition to adaptations for hibernation.  4.2.1. Hamsters require I  NaP  to set respiratory rhythm  Using the in situ preparation, eliminating I  NaP  by applying riluzole in age-matched rats and  hamsters had different concentration-dependent effects. No hamster preparations continued generating fictive breathing past 14 pM riluzole while weaned rats continued fictive breathing until 20 pM riluzole. Application of riluzole resulted in a profound decrease in phrenic burst frequency of hamster preparations at very low concentrations (Figure 3-2A). The concentrations that yielded a 50% decrease in phrenic burst frequency (1-2 pM) would have specifically blocked only I  NaP  (Urbani 6k Belluzzi, 2000; Del Negro et al, 2005). Frequency in weaned rats did not  change significantly from baseline values at these concentrations. That riluzole affects frequency in hamsters, not rats, was observed in the isolated brainstem-spinal cord preparation in early postnatal development (0-4 PND) (Marshall, 2005), suggesting that the difference exists at birth.  42  This response of the hamsters is different than all existing reports of riluzole application in mice and rats. Because riluzole had no effect on burst frequency in rat and mouse brainstem transverse slices, in situ preparations or intact rats, I  NaP  is not considered necessary for respiratory  rhythm generation (Del Negro et al, 2005)(Paton et al, 2006). When rhythm has been abolished, the effect had been attributed to the non-specific actions of riluzole at the high concentrations used ( 1 0 - 2 0 pM) (Del Negro et al., 2005). The response of the in situ rat preparations in this study is consistent with the present literature because frequency did not change significantly with increasing riluzole. However, our data differ for hamsters suggesting that the C R G is being affected by the application of riluzole and that in hamsters I  NaP  plays an important role in neurons  that set respiratory rhythm. In contrast with the drastic effect of eliminating I , FFA application had no significant NaP  effect on frequency in either hamsters or rats (Figure 3-5A) until concentrations >25 pM. Between 25 - 100 pM, FFA application resulted in the abolition of fictive breathing in most of the preparations through a decrease in frequency. At concentrations >25 pM, FFA will block not only ICAN but also will interfere with various Ca , CI" and K* currents and gap junctions (described in 2+  section 4.1) (White & Aylwin, 1990; Farrugia et al, 1993; Ottolia & Toro, 1994; Li et al, 1998; Harks et al, 2001; Stumpff et al, 2001; Srinivas &. Spray, 2003). Application of FFA less than 25 pM most likely led to decreased excitability of the neuronal network which resulted in the decline in net motor output but had less effect on frequency in both rats and hamsters. The minor effect of FFA on frequency in our study is in accord with current literature that also does not report FFA having a frequency effect (Pena et al, 2004; Pace et al, 2007a). When riluzole and FFA were coapplied, phrenic burst frequency in hamsters decreased significantly at low doses while the frequency in rat preparations was unchanged (Figure 3-9A). However, when compared to the decrease in frequency in response to riluzole alone, frequency in hamster preparations did not decline as much during coapplication at the corresponding concentrations. For instance, 50% of hamster preparations were able to continue fictive breathing during the coapplication of riluzole and FFA. The last concentration of riluzole in the combination is 20 pM. When riluzole was applied individually, no hamster preparations continued fictive breathing past 14 pM riluzole. This result suggests that FFA may mitigate some 43  of the effects of eliminating I  NaP  in hamsters. FFA is known to block gap junctions (Harks et al,  2001; Srinivas &. Spray, 2003) and blockade of gap junctions in the in situ preparation has been shown to lead to increased phrenic burst frequency (Solomon et al, 2003), which may be one mechanism by which FFA could negate some of the effects of eliminating I  NaP  on frequency. Also,  at the majority of the concentrations applied (2.5 - 15 pM), FFA would stimulate large conductance Ca -activated K channels (between 5 - 1 0 pM) (Kochetkov et al, 2000), which may z+  4  counteract the effect of riluzole in decreasing neuronal excitability, and result in less of a decline in frequency. In contrast with the hamster, coapplication of riluzole and FFA in rat preparations led to a greater decrease in fictive breathing: only 30% of weaned rats continued generating phrenic bursts, whereas during riluzole alone, all weaned rats continued bursting at these concentrations (Table 3-4). In addition, in weaned rats, 12 pM riluzole only resulted in a 10% reduction in frequency; however when 12 pM riluzole was coapplied with 10 pM FFA, frequency was reduced by 34%. The ability of the coapplication of riluzole and FFA to reduce the frequency of the respiratory rhythm in our study corresponds with reports in brainstem transverse slice preparations and intact animals where respiratory motor output was abolished when riluzole and FFA were coapplied but not when applied individually (Pena et al, 2004; Del Negro et al., 2005; Pace et al, 2007b; Pena &. Aguileta, 2007). However, in these studies, the concentration of FFA used was very high (100 - 500 pM), and motor output declined due to falling amplitude, not frequency (Pena et al, 2004; Del Negro et al, 2005; Pace et al, 2007b; Pena & Aguileta, 2007). Therefore, the eliminating I  NaP  and I ^ N affected the neurons that set respiratory rhythm in the  C R G in hamsters, but not in rats. 4.2.2. Respiratory burst amplitude is affected in rats more than hamsters by the elimination of both I The elimination of only I  NaP  NaP  and 1^^  by the application of riluzole resulted in a significant decrease  in burst amplitude in hamsters at low concentrations of riluzole and in rats at higher concentrations (Figure 3-2B). I  NaP  is important in promoting repetitive bursting in respiratory  neurons (Crill, 1996; Del Negro et al, 2002b). Blocking I , and therefore repetitive bursting, NaP  may impair the overall network ability to generate motor output and lead to an attenuation of the 44  amplitude of phrenic nerve discharges. Previous studies also show that application of riluzole reduced the amplitude of motor output from X I I motor neurons (Del Negro et al, 2005)(Del Negro et al, 2002b).  Riluzole decreased excitatory post-synaptic potentials and abolished  repetitive firing (but not action potential generation) which had the net effect of reducing the excitability of X I I motoneurons (Bellingham, 2006). The systemic application of riluzole in the present study would lead to similar effects on phrenic motor neurons and reduce neuronal excitability in the phrenic motor nucleus, accounting for our observed reduction in amplitude (Figure 3-2B). Rat preparations, but not hamster preparations, significantly reduced amplitude in response to FFA concentrations <25 pM (Figure 3-5B). The fall in amplitude in rats likely was caused by the reduced excitability of the respiratory network that results from the elimination of ICAN  and all the other currents that FFA affects. Hamster preparations showed little response to  FFA until higher concentrations. A response may have been observed if larger concentrations of 1  FFA had been applied as was seen in weaned rats at concentrations >25 pM (Figure 3-7). Unfortunately, applying higher concentrations of FFA confound the interpretation of results due to the multiple effects of FFA at high doses (White & Aylwin, 1990; Farrugia et al, 1993; Ottolia & Toro, 1994; Li et al, 1998; Harks et al, 2001; Stumpff et al, 2001; Srinivas & Spray, 2003). Coapplication of riluzole and FFA resulted in a significant decrease in amplitude in both hamster and rat preparations (Figure 3-9B). Similar to the frequency response, amplitude in hamsters was affected less by the coapplication of riluzole and FFA than by riluzole alone. Amplitude was reduced to 6% of baseline when riluzole was applied alone but only 55% when riluzole and FFA were coapplied (Figure 3-2B 6k Figure 3-9B), suggesting that FFA may mitigate the reduction in amplitude. Our results correspond with previous reports that show that coapplication of riluzole and FFA reduced or abolished burst amplitude in rats (Pena et al, 2004; Del Negro et al, 2005; Pace et al, 2007b). Due to their functions in promoting repetitive bursting (I p) and producing the inspiratory drive potential Na  (ICAN)>  eliminating I  NAP  and  ICAN  would lower  the excitability of respiratory neurons (with or without intrinsic bursting properties) and impair the ability of the respiratory network to generate output to the respiratory motor neurons.  45  4.2.3. Hamster neural ventilation decreases more than rat upon elimination of both I  NaP  and LJAJJ  Eliminating I  NaP  alone in hamster in situ preparations resulted in a significant decrease in  neural ventilation (nV ) at lower concentrations than in rat preparations (Figure 3-2C). The E  decrease in nV in hamsters was produced by the decrease in both frequency and amplitude; in F  rats, the decrease in nV  E  was caused by the decrease in amplitude. That respiratory rhythm  generation in hamsters was very sensitive to elimination of I  NaP  suggests that the hamster  respiratory network may be configured differently than the network in rats or that I  NaP  is more  dominant among the mechanisms responsible for rhythm generation in hamsters. The low concentrations which elicited a change in frequency and the C V N validation recordings together suggest that this result is not a side effect of the non-specific actions of riluzole or instability of hamster in situ preparations. Applying FFA resulted in a significant decrease in nV  E  in rats while nV  E  in hamster  preparations remained around baseline values (Figure 3-5C). In both hamster and rats, any drop in nV due to FFA was caused by the observed decrease in amplitude (Figure 3-5B). E  After applying riluzole and FFA to rat in situ preparations individually, concentrations of each drug were chosen for the coapplication of riluzole and FFA in order to give progressively larger inhibition of nV . Applying these drug combinations resulted in significantly decreased E  nV in both hamster and rat preparations (Figure 3-9C). We calculated expected values for nV E  E  (Figure 3-10) on the assumption that the effect of the drugs would be additive. Comparing the expected values to the actual nV values obtained showed that, in the in situ preparation, nV E  E  declined more than was expected if the effects of riluzole and FFA were simply additive. This difference between expected and actual nV values also highlights the drastic difference between E  rats and hamsters to the elimination of I  NaP  and I  fictive breathing following the elimination of I  NaP  CAN  . More hamster preparations still exhibited  and  o n l  Y 30% of weaned rats but 86% of  hamsters continued generating phrenic bursts at the highest concentration (Table 3-4). Hamsters decreased burst frequency more but defended amplitude better than rats, which decreased  46  amplitude more than frequency; however, both rats and hamsters suppressed nV to the same E  extent (Figure 3-9). Considering the number of studies that have coapplied riluzole and FFA to rats and mice and observed little change in respiratory rhythm (Del Negro et al, 2002b; Pena et al, 2004; Del Negro et al, 2005; Pace et al, 2007b, a; Pena 6k Aguileta, 2007), the stark difference in the response to hamsters to elimination of I  NaP  and ICAN supports our hypothesis that hamsters are  more reliant on these currents for rhythm generation, reflecting a fundamental difference in the CRG between rats and hamsters. 4.2.4. Increasing drive only rescues 'breathing' in rats If the elimination of I  NaP  and l  CAN  reduces the excitability of the respiratory network and  removes a source of drive to the CRG thereby decreasing nV in rat and hamster preparations, E  increasing the proportion of C 0  2  in the perfusate of the in situ preparation to provide an alternate  source of drive to the C R G should counteract the effect of the elimination of I  NaP  or ICAN-  Increasing the proportion of C O from 5% (Pco2 ~ 42 Torr) to 7% (Pco2 ~ 52 Torr) in the z  perfusate resulted in a large but not significant increase in the frequency of phrenic bursting in weaned rat preparations but no change in hamster preparations (Table 3-6). As riluzole and FFA were applied in hamsters, frequency values at 7% C 0 tended to be greater, but not significantly, 2  than values at 5% C 0 (Figure 3-12C). In intact hamsters exposed to a 5% hypercapnic 2  environment, frequency decreased, which does not correspond with our observed increase (Walker et al, 1985). However, amplitude tended to be greater in 7% C 0 preparations (Figure 2  3-13C) which agrees with the increase in amplitude seen in intact hypercapnic hamsters (Walker et al, 1985). Overall ventilation increased in intact hamsters in response to hypercapnia (5%) while in the in situ preparation, nV was not different in 7% C 0 preparations than in 5% C 0 E  2  2  preparations (Figure 3-14C). In contrast, weaned rat preparations significantly increased nV  E  (Figure 3-14C) in response to 7% C 0 by increasing frequency (Figure 3-12C) and significantly 2  increasing amplitude (Figure 3-13C). These results agree with previous studies in rat in situ preparations which reported that increasing perfusate P o2 increased phrenic burst frequency (St.C  John et al, 2007) and increased amplitude resulting in an overall increase in neural ventilation  47  (Day 6k Wilson, 2005). Ventilation also increased in intact rats exposed to hypercapnia (Walker et al, 1985)(Cragg 6k Drysdale, 1983). In rats, providing an alternate source of drive to the C R G through activation of chemoreceptors recovered some of lost respiratory output due to application of riluzole and FFA, which is consistent with the primary effects of hypercapnia on amplitude. Increasing C 0 in the 2  hamster in situ preparations did not change total ventilation or the response to eliminating I and  ICAN;  NAP  therefore, chemoreceptor drive to the respiratory network was not able to reduce the  effects of eliminating l  N a P  effects on rhythm that I  NAP  and I AN- These results suggest that increasing drive cannot replace the C  and I N provide. The estimated P 2 of the 7% C O is 52 Torr; in vivo C A  CQ  z  hamster P032 is reported to be 52 Torr and does not change during exposure to a 5% hypercapnic environment (Pco2  =  55 Torr) (Walker et al, 1985). Since hamsters have a blunted hypercapnic  ventilatory response, the change in P  C D 2  in the perfusate may not have been enough to stimulate a  chemosensory response in in situ hamster preparations; therefore, we cannot conclude if increasing drive is able to recover respiratory motor output in hamsters, as it does in rats.  4.3. Ontogenetic differences in I  N A P  and  ICAN  A critical developmental window exists between 16 - 18 P N D in rats where the ability to spontaneously recover from hypothermia-induced respiratory arrest is lost. W e hypothesize that I  NaP  and  and I  C A N  ICAN  a  r  e  involved with initiating recovery and that reductions in the relative roles of I  NAP  in the respiratory C R G result in this developmental transition.  4.3.1. Respiratory rhythm in young rats is more sensitive to application riluzole and FFA Application of riluzole began reducing phrenic burst frequency in young rats significantly from baseline at 10 p M while phrenic burst frequency in weaned rat preparations remained at baseline levels (Figure 3-2A). This result is inconsistent with most published studies that report either that riluzole does not affect respiratory rhythm or that any effect that occurs is a result of riluzole blocking other currents in addition to I  NaP  (Del Negro et al, 2002b)(Del Negro et al,  2005)(Paton et al, 2006; Pena 6k Aguileta, 2007; St.-John et al, 2007). One study reports that riluzole affects frequency in rats in a medullary in situ preparation (similar to the in situ 48  preparation but the pons is removed). However this data showed a decrease in amplitude only (not frequency) before motor output was abolished (Ramirez 6k Viemari, 2005). Based on the literature, which shows that I  NaP  is eliminated by riluzole with E C  50  = 3 pM in brainstem  transverse slices, the change in frequency observed in the young rat preparations in the current study at 10 pM is most likely a result of the actions of riluzole on other channels in addition to blockade of I  NaP  in young rats (Del Negro et al, 2005). The decline in frequency occurred £10 pM  riluzole in young rats leading to a significant difference between the two ages of rats suggesting that riluzole (all interactions) have more of an effect in young rats. FFA application eliminated fictive breathing in young rat preparations as concentration increased so that only 60% of preparations remained active after the last concentration (Table 3-3). Applying FFA at a concentration of 25 pM had little effect on frequency and, therefore, respiratory rhythm generation in rats (Figure 3-5B). However, for rat preparations that did not continue to the last concentration of FFA (25 pM), respiratory rhythm was abolished due to amplitude reaching zero before frequency. Increasing FFA concentration to 100 pM resulted in a decrease in frequency however at this concentration the other multiple actions of FFA will be exerting non-specific effects on the expression of the respiratory rhythm (White 6k Aylwin, 1990; Farrugia et al, 1993; Ottolia 6k Toro, 1994; Li et al, 1998; Harks et al., 2001; Stumpff et al., 2001; Srinivas 6k Spray, 2003). When riluzole and FFA are added in combination, no young rats continued bursting at the highest concentration (20 pM RIL + 15 pM FFA) whereas for both riluzole and FFA individually, young rat preparations continued generating bursts to the last concentration. The coapplication of riluzole and FFA also abolished phrenic motor output in all but 30% of weaned rat preparations (Table 3-4) while all weaned rat preparations survived the individual applications of riluzole or FFA. Coapplication of riluzole and FFA during early post-natal development (in brainstem transverse slices) as well as at 9 - 13 PND (in in vivo mice) abolished respiratory rhythm, which corresponds to our data (Del Negro et al, 2005; Pena 6k Aguileta, 2007). Phrenic burst frequency was affected in both ages of rats; however, the decrease in young rats was only significantly different from weaned rats at the highest concentration (>10 pM riluzole and 8 pM FFA) (Figure 3-9A), suggesting a developmental change occurs between 14 - 23 PND in the 49  sensitivity to riluzole and FFA in rats. Our data are consistent with the existence of a hypothesized developmental window where autoresuscitation ability is lost in non-hibernating species but the question of whether I  NaP  and  ICAN  mediate autoresuscitation requires direct testing.  4.3.2. Young rats are more dependent on I  NaP  and I  CAN  than weaned rats for producing  motor output Young rats significantly decreased amplitude at concentrations £12 pM riluzole while weaned rats maintained amplitude closer to baseline values resulting in a significant difference between the ages (Figure 3-2B). Riluzole is reported to reduce amplitude in rats and mice in in situ and brainstem transverse slice preparations suggesting that riluzole affects components of the respiratory network that are important for generating motor output (more than setting rhythm) (Pena et al, 2004; Del Negro et al, 2005; Ramirez & Viemari, 2005). Both young and weaned rats decreased amplitude to the same extent during FFA application up to 25 pM (Figure 3-5B). In weaned rat preparations, applying FFA up to 100 pM reduced amplitude to zero in most preparations (Figure 3-7). ICAN is responsible for the depolarization of the membrane of inspiratory neurons called the inspiratory drive potential, which facilitates neuronal bursting (Del Negro et al, 2005)(Pena et al, 2004; Pace et al, 2007a). Therefore, attenuation of ICAN would reduce the inspiratory drive potential and excitability of respiratory neurons, leading to decreased drive to the motor neurons and smaller amplitude. However, in brainstem transverse slice preparations, when inspiratory drive potential was eliminated by FFA, very little change was seen in the XII motor output (Pace et al, 2007a); therefore, the observed decrease in motor output in the current study cannot only be attributed to attenuation of  ICAN-  At doses between 25 - 100 pM, FFA will interfere with gap junctions and  various C a \ K and CI" currents and have the overall affect of reducing excitability of the 2  +  respiratory network (as discussed in section 4.1) (White 6k Aylwin, 1990; Farrugia et al, 1993; Ottolia 6k Toro, 1994; Li et al, 1998; Harks et al, 2001; Stumpff et al, 2001; Srinivas 6k Spray, 2003), which would also result in reducing amplitude. Coapplication of riluzole and FFA significantly reduced amplitude in young and weaned rats similarly so that the age groups were not significantly different from each other (Figure 3-9B). Our results are supported by reports from rat and mouse brainstem transverse slice studies and 50  studies using intact awake mice which abolished inspiratory rhythm by coapplication of riluzole and FFA by mostly affecting the amplitude of the motor output (Pena & Aguileta, 2007)(Pena et al, 2004; Del Negro et al, 2005). With our systemic method of applying riluzole and FFA, the drugs would have access to all regions of the brain, so the change in motor output we saw likely were caused by the decreased neuronal excitability caused by the action of the drugs at multiple sites. 4.3.3. Ventilation in young rats declined more than in weaned rats All our weaned rat preparations survived the elimination of I  NAP  through the application of  riluzole while the majority of young rat preparations did not (Table 3-3). nV was decreased more E  in young rats above 8 pM so the response of young rats was significantly different from the response of weaned rats (Figure 3-2C), suggesting that young rats are more sensitive to the elimination of I  NAP  than weaned rats and may rely more on I  NAP  for maintenance of rhythm  generation. Both rat groups significantly decreased nV after application of FFA (0.25 - 25 pM) so E  that no significant difference existed between the age groups (Figure 3-5C). When 25 - 100 pM FFA was applied to weaned rats, amplitude, frequency, and therefore n V , all declined (Figure E  3-7). Only one third of weaned rat preparations continued generating respiratory motor output past 50 pM FFA; the multiple effects of FFA at these concentrations could result in a decrease in frequency and/or amplitude of phrenic bursts due to reduced network excitability. During coapplication of riluzole and FFA, nV decreased significantly in both age groups E  so that the groups were not significantly different (Figure 3-9C). The drop in n V was due to the E  decreases in both amplitude and frequency and was much greater than expected if the drugs had simply additive effects. Despite differences in the components, no significant difference in total  n V existed between the age groups when I E  NAP  and I C A were eliminated together. N  4.3.4. Increasing drive only rescues 'breathing' in weaned, not young, rats Burst frequency (Figure 3-12A, B) increased in both weaned and young rats upon increasing the proportion of C 0 from 5% to 7% in the perfusate. Amplitude (Figure 3-13A, B) 2  51  increased in 7% C 0 more in weaned rats than in young rats. Therefore, neural ventilation 2  (Figure 3-14A, B) increased in response to 7% C 0 to a greater extent in weaned rats than in 2  young rats. These results agree with observed hypercapnic ventilatory response in intact rats (Walker et al, 1985)(Cragg 6k Drysdale, 1983) and rat in situ preparations (St.-John et al, 2007) (Day 6k Wilson, 2005). In weaned rats, nV was greater at 7% C 0 than 5% C 0 , and it significantly dropped as E  2  2  drug concentrations increased but the significant difference between 7% C 0 and 5% C 0 2  2  preparations was maintained until the final concentration. In contrast, nV in young rats also E  increased in 7% C 0 preparations but decreased significantly to match the values of 5% C 0 2  2  preparations at higher drug concentrations. Hypercapnia seemed to ameliorate the effects of the drug combination on the amplitude in weaned rats; young rat burst amplitude decreased the same way in normal or high drive preparations. These results suggest that providing drive from chemoreceptors was able to reduce the degree to which amplitude and frequency were reduced but to different extents in young and weaned rats. The lack of significant response from the young rats to the increase in C 0 may to due to 2  incomplete development of the hypercapnic ventilatory response. The hypercapnic ventilatory response in rats undergoes developmental changes in the first two weeks of life. In the first five days, response to hypercapnia is large and declines to a nadir at 8 PND. At 12 PND, ventilatory response just starts to increase from the nadir toward adult levels (Putnam et al, 2005). Therefore at the ages used in the current study, the young rats (12 - 14 PND) are at their lowest level of responsiveness to C 0 while the weaned rats have a fully developed hypercapnic ventilatory 2  response. Our results from weaned rats agree with brainstem transverse slice experiments where coapplication of RIL and FFA on rat and mouse slices abolished respiratory rhythm at 20 pM riluzole and 100 pM FFA (both at cellular and XII motor neuron activity) (Del Negro et al, 2005). This effect was counteracted by increasing membrane excitability by application of substance P so the investigators concluded that I  NaP  and IQ^N contribute to rhythm generation simply by  increasing excitability and promoting inspiratory burst generation in all PBC neurons (Del Negro 52  et al, 2005). Our results confirm that riluzole and FFA reduce network excitability which can be rescued by substance P (Del Negro et al, 2005) or increased chemoreceptor drive (Figure 3-14B).  4.4.Speculations We observe that respiratory rhythm (frequency) in hamsters is more dependent on I  NaP  than in rats. Our results from young rats also suggest that younger rats may be more dependent on I  NaP  and ICAN for rhythm generation than weaned rats. Although we do not test it directly here, our  results are consistent with the 'hibernator as neonate' hypothesis. This hypothesis is derived from the observation that adult hibernators share a number of characteristics with neonates of all mammals (such as cold and hypoxia tolerance) (Harris et al, 2004). In this case, reliance on I  NaP  (and ICAN) f ° rhythm generation may be a neonatal characteristic retained by hamsters into r  adulthood. The ability to hibernate is not the only way in which hamsters and rats differ. Hamsters are fossorial species with well developed adaptations to living in hypoxic, hypercapnic burrows while rats are a more generalist species with fewer specific adaptations to fossorial lifestyles (Walker et al, 1985; Frappell 6k Mortola, 1994). Non-pacemaker and IcAN-mediated bursting neurons are proposed to be silent during hypoxia (Ramirez 6k Viemari, 2005). Therefore, living in chronic hypoxic burrows may have resulted in a reconfiguration of the hamster C R G to one that relies more on I -mediated bursting neurons (compared to the rat) since expression of ICAN  m  NaP  a  Y  have been reduced as hamsters adapted to their fossorial lifestyle. Our results that show that hamsters are very sensitive to riluzole application are consistent with a C R G that is more dependent on I -mediated mechanisms of rhythm generation. In addition, in hamsters P NaP  greater and P  0 2  C 0 2  is  is less than that of rats at normoxia and normocapnia; therefore, central  chemoreceptors may be active more in hamsters resulting in greater chemoreceptor drive to the PBC (Mulkey et al, 2004). The increased tonic excitatory drive from central chemoreceptors could compensate for the lost source of drive from the reduced expression of ICAN- Therefore, the reliance of hamsters of I  NaP  for respiratory rhythm generation may stem from adaptations to a  fossorial life.  53  4.5. Conclusions Applying riluzole and FFA to in situ rat and hamster preparations resulted in a large decrease in the frequency of phrenic nerve bursting in hamsters in response to the elimination of I . Hamsters rely more on I -mediated mechanisms to set respiratory rhythm than rats. NAP  NaP  Therefore, a fundamental difference exists between adult rat and hamster respiratory rhythm generation. This species difference highlights the need to apply the models of rhythm generation based on rats and mice carefully across mammalian species. When riluzole and FFA were applied to two age groups of rats, young rats (12 - 14 PND) tended to decrease frequency more than weaned rats (>23 PND); therefore, younger rats may be more reliant than weaned rats on I  NAP  and IcAN-mediated mechanisms of rhythm generation. Our  data are consistent with the hypothesis that a developmental change occurs in the relative roles of I  NAP  and  ICAN  in the C R G of mammals.  Both hamsters and young rats showed a greater reliance on I  NAP  and  ICAN  f ° rhythm r  generation than weaned rats; both hamsters and young rats also can autoresuscitate. This correlation is consistent with the suggestion that I  NAP  and IcAN-mediated bursting neurons may  facilitate recovery from hypothermic respiratory arrest.  54  5. 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Respir Physiol Neurobiol 149, 83-98.  60  6. Appendix Table 6-1: Tj and T values for young rats, weaned rats and weaned hamsters in the riluzole trials Asterisk (*) denotes significance from baseline. E  [RIL] (uM) 0.0 0.2 1.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0  Young rats 1.00 ± 0.00 1.04 ± 0.02 0.93+0.02 0.88 ± 0.01 0.94 ± 0.03 0.92 ± 0.06 0.92 ± 0.08 1.02 ±0.17 1.11 ±0.31 1.46 ±0.67 1.46 ±0.70 1.51 ±0.71 1.30 ±0.37  T Weaned rats 1.00 ± 0.00 0.98 ± 0.02 0.96 ± 0.02 0.97 ± 0.04 0.84 ± 0.04 0.83 ± 0.05 0.94 ± 0.07 1.04 ± 0.08 1.15 ±0.10 1.20 ± 0.09 1.21 ±0.14 1.30 ±0.12 1.32 ±0.12  T  Hamsters 1.00 ±0.00 1.12 ±0.06 1.19 ±0.07 1.18 ±0.16 1.15 ±0.08 1.10 ±0.13 1.29 ±0.17 1.05 ±0.22 1.55 1.42  Young rats 1.00 ± 0.00 1.02 ± 0.02 1.07 ± 0.06 1.06 ± 0.08 1.27 ±0.21 1.75 ±0.40 2.16 ±0.62 2.57 ±0.74 3.31 ± 1.85 1.58 ±0.33 1.91 ±0.30 2.84 ± 0.63 4.48 ± 2.06*  E  Weaned rats 1.00 ± 0.00 1.07 ± 0.02 1.07 ± 0.04 1.09 ± 0.06 1.09 ± 0.08 1.15 ±0.11 1.09 ±0.15 1.19 ±0.22 1.30 ± 0.27 1.32 ±0.26 1.43 ±0.28 1.65 ±0.41 1.72 ±0.32  Hamsters 1.00 ± 0.00 1.38 ±0.18 1.50 ±0.23 1.89 ±0.31 2.04 ± 0.46 2.94 ± 0.45 2.98 ± 1.81 2.66 ± 0.63 5.25 7.15  Table 6-2: T, and T values for young rats, weaned rats and weaned hamsters in the FFA trials. E  [FFA] (uM) 0.00 0.25 1.25 2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00  Young rats 1.00 ± 0.00 1.10 ±0.16 0.98 ± 0.05 0.87 ± 0.05 0.81 ±0.07 0.81 ±0.06 0.70 ± 0.03 0.73 ± 0.02 0.63 ± 0.01 0.61 ±0.03 0.64 ± 0.04 0.64 ± 0.03 0.63 ± 0.04  T, Weaned rats 1.00 ±0.00 1.05 ±0.05 1.02 ±0.01 0.95 ± 0.03 1.03 ± 0.04 1.08 ± 0.09 1.19 ±0.22 1.24 ±0.29 1.24 ±0.33 1.02 ±0.13 1.03 ±0.14 0.91 ± 0.09 0.83 ± 0.15  T Weaned rats 1.00 ± 0.00 0.99 ±0.11 0.95 ± 0.08 0.97 ±0.14 0.95+0.12 0.92 ±0.10 0.99 ± 0.13 1.29 ±0.39 1.04 ± 0.09 1.47 ± 0.42 1.11 ±0.11 1.81+0.63 2.12 ± 1.23 E  Hamsters 1.00 ±0.00 0.97 ± 0.03 1.07 ± 0.07 0.97 ± 0.08 0.89 ±0.12 0.87 ±0.11 0.62 ± 0.06 0.72 ±0.06 0.92 ± 0.08 0.87 ±0.10 0.88 ± 0.09 0.78 ±0.16 0.66 ±0.16  Young rats 1.00 ± 0.00 1.04 ± 0.07 0.98 ± 0.07 1.00 ± 0.06 1.02 ±0.08 1.34 ± 0.34 0.80 ±0.12 0.89 ± 0.20 1.23 ± 0.68 0.52 ± 0.04 0.51 ±0.04 0.37 ± 0.03 0.35 ± 0.03  Hamsters 1.00 ± 0.00 1.01 ±0.07 0.97 ± 0.05 0.91 ± 0.09 0.87 ±0.10 0.88 ±0.12 0.87 ±0.10 0.96 ±0.15 0.90 ±0.14 0.75 ±0.10 0.91 ±0.22 0.76 ±0.18 1.00 ± 0.32  61  Table 6-3: Ti and T data for young rats, weaned rats and weaned hamsters in the trials with coapplication of riluzole and FFA. . E  [RILMFFA]  T  T,  E  Young rats  Weaned rats  Hamsters  Young rats  Weaned rats  Hamsters  OuM  1.00 ± 0.00  1.00+0.00  1.00 ± 0 . 0 0  1.00 ± 0.00  1.00 ± 0.00  1.00 ± 0.00  6uM/2.5uM  0.89 ± 0.03  0.84 ± 0.02  1.11 ± 0 . 1 2  1.34 ± 0 . 1 1  1.03 ± 0 . 0 8  1.85 ± 0.35  7.5uM/5uM  0.92 ± 0.02  0.81 ± 0 . 0 2  1.19 ± 0 . 1 9  1.54 ± 0 . 1 6  1.11 ± 0 . 0 9  3.37 ± 0 . 7 5  1.24 ± 0 . 1 9  2.14 ± 0 . 3 3  1.32 ± 0 . 1 8  15.46 ± 6.32  9uM/6uM  1.04 ± 0 . 0 6  0.99 ± 0.06  10UM/8UM  1.15 ± 0 . 0 9  1.01 ± 0.07  1.26 ± 0 . 2 6  2.33 ± 0.38  1.52 ± 0 . 2 3  37.42 ± 2 5 . 8  12uM/10uM  1.09 ± 0.09  1.12 ± 0 . 0 5  0.99 ± 0.24  2.62 ± 0 . 7 5  2.09 ± 0.56  28.30 ± 17.8  1.30 ± 0 . 3 0  1.33 ± 0 . 2 7  3.56 ± 1.02  3.25 ± 0.96  20uM/15uM  Table 6-4: T[ and T values for young rats, weaned rats and weaned hamsters in the trials with coapplication of riluzole and FFA E  [RIL]/[FFA]  T,  TE  Young rats  Weaned rats  Hamsters  Young rats  Weaned rats  Hamsters  OuM  1.10 ± 0 . 0 8  0.93 ± 0.07  1.01 ± 0 . 1 9  1.16 ± 0 . 0 7  1.29 ± 0 . 2 3  0.94 ± 0 . 2 2  6uM/2.5uM  1.07 ± 0.09  0.92 ± 0.06  1.12 ± 0 . 2 2  1.28 ± 0 . 1 8  1.59 ± 0 . 4 7  2.10 ± 0 . 6 6  1.72 ± 0 . 5 5  4.06 ± 2.24  7.5uM/5uM  1.28 ± 0 . 0 7  1.06 ± 0 . 1 0  1.90 ± 0 . 8 5  1.95 ± 0 . 5 2  9uM/6uM  1.17 ± 0 . 0 8  1.11 ± 0 . 1 1  3.01 ± 1.62  1.31 ± 0 . 1 3  1.80 ± 0 . 5 6  5.64 ± 3 . 3 0  10uM/8uM  1.21 ± 0 . 0 9  1.10 ± 0 . 1 4  5.26 ± 2 . 7 6  1.48 ± 0 . 1 8  2.06 ± 0 . 7 1  17.27 ± 14.89  12uM/10uM  1.25 ± 0 . 1 3  1.21 ± 0 . 1 6  4.34 ± 2 . 1 6  2.04 ± 0.32  2.17 ± 0 . 7 4  6.44 ± 5.03  20uM/15uM  1.18 ± 0 . 1 4  1.13 ± 0 . 1 7  1.47 ± 0 . 3 9  5.38 ± 4 . 5 6  3.44 ± 1.00  4.19 ± 1 . 3 1  62  

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