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

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SPECIES AND 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 (INAP) and Ca2*-activated non-selective cation 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 IN A P and I C A N contribution to respiratory rhythm generation change as a rat ages. We applied riluzole (IN A P 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 IN A P and I C A N 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 CRG 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 IN A P and for respiratory rhythm generation are consistent with the hypothesis that a developmental change occurs in the CRG of rats during maturation. Increasing the proportion of C 0 2 that the preparations were exposed to increased neural ventilation in weaned rats suggesting that IN a P and ICAN provide a source of excitatory drive 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, 1.2. Phylogeny and respiratory rhythm generation 4 1.2.1. Do phylogenetic differences exist in the roles of IN a P and ICAN in respiratory rhythm generation? 4 1.3. Ontogeny and respiratory rhythm generation 5 1.3.1. Do ontogenetic differences exist in the roles of IN a P and I C A N in respiratory rhythm generation? 5 2. Methods 8 2.1. In situ working heart-brainstem preparation 8 2.2. Experimental protocol 9 2.3. Data and statistical analysis '. 10 3. Results 16 3.1. Riluzole application 16 3.2. Flufenamic acid application 18 3.3. Riluzole and FFA coapplication 19 3.3.1. Coapplication at 5% C O z 20 3.3.2. Coapplication at 7% C 0 2 : 21 4. Discussion 40 4.1. Technical considerations 40 4.2. Phylogenetic differences in IN a P and ICAN contribution to rhythm generation 42 4.2.1. Hamsters required IN a P to set respiratory rhythm 42 4.2.2. Respiratory burst amplitude is affected in rats more than hamsters by the elimination of IN a P and ICAN 4 4 4.2.3. Hamster neural ventilation decreases more than rat upon elimination of IN a P and ICAN46 4.2.4. Increasing drive only rescues 'breathing' in rats 47 4.3. Ontogenetic differences in IN a P and I C A N 4 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 IN a P and ICAN m a n weaned rats for producing motor output 50 4.3.3. Ventilation in young rats declined more than in weaned rats 51 iii 4.3.4. Increasing drive only rescues 'breathing' in weaned, not young, rats 51 4.4. Speculations 53 4.5. Conclusions 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 2 producing respiratory rhythm during coapplication of riluzole and FFA 37 Table 3-6: Alterations in phrenic nerve burst frequency, T[ and T E of young rat, weaned rat and weaned hamster preparations exposed to 5% C 0 2 or 7% C 0 2 . Values are expressed as means±standard error. N=number of preparations 37 Table 6-1: T, and T E values for young rats, weaned rats and weaned hamsters in the riluzole trials. Asterisk (*) denotes significance from baseline 61 Table 6-2: T, and T E values for young rats, weaned fats and weaned hamsters in the FFA trials. ..61 Table 6-3: T[ and T E data for young rats, weaned rats and weaned hamsters in the trials with coapplication of riluzole and FFA 62 Table 64: T[ and T E values for young rats, weaned rats and weaned hamsters in the trials with coapplication of riluzole and FFA 62 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 (nVE, C), corrected for vehicle controls, in response to increasing concentrations of riluzole 26 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 (nVE, C), corrected for vehicle controls, in response to increasing concentrations of FFA 30 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 (nVE) all declined as FFA concentration increased in weaned rat preparations (n=6) 32 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 (nVE, C) corrected for vehicle controls, in response to increasing concentrations of riluzole + FFA 34 Figure 3-10: Expected values for neural ventilation (nVE) based on additive action of riluzole and FFA compared to the actual nVE for young rats (A), weaned rats (B) and weaned hamsters (C) 35 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 z or 7% C 0 2 and coapplication of riluzole and FFA 38 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 2 or 7% C 0 2 and coapplication of riluzole and FFA 38 Figure 3-14: Changes in neural ventilation (nVE), corrected for vehicle controls, for young rat (A), weaned rat (B) and hamster (C) preparations exposed to perfusate equilibrated with 5% C 0 2 or 7% C O z and coapplication of riluzole and FFA 39 vii List of abbreviations BotC Botzinger Complex CRG Central rhythm generator CVN Cervical vagus nerve cVRG Caudal ventral respiratory group FFA Flufenamic acid I C A N Ca2+-activated non-selective cation current IK+-LEAK K + leak current IN a P Persistent Na+ current IN a T Transient Na+ current mV Millivolts YI VE 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, Bi l l Milsom, for his expertise and support and for deliberately creating a familial environment in our lab. Thank you to my committee members Vanessa Auld 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 Missouri-Columbia 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 Don 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; Hi l l , 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 PND; 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 PBC has been postulated to be the site of inspiratory rhythm generation in the brainstem. The PBC 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 PBC 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 voltage-dependent persistent sodium current (INaP) were proposed to be necessary to set respiratory rhythm (Smith et al, 1991; Johnson et al, 1994; Del Negro et al, 2002a; Del Negro et al, 2002b). I N a P is active at more negative membrane potentials than other currents: I N a P is active between -60 and -40 mV, transient sodium current (INaX) is active at -34 mV (Richter & Spyer, 2001)(Butera Jr. et al, 1999)(Urbani 6k Belluzzi, 2000)(Alzheimer et al, 1993). I N a P 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 PBC interneurons (Ptak et al, 2005). I N a P facilitates respiratory rhythm generation by promoting plateau potentials and repetitive firing and by increasing neuron excitability (as depicted in Figure 1-2). I N a P is activated at -60 mV and depolarizes the membrane to the activation threshold for I N a X (Figure 1-2BCD), 2 leading to voltage-dependent bursting (Figure 1-2B©) (Feldman 6k Del Negro, 2006). During the action potential I N A T becomes inactivated rapidly; however, due to the slow inactivation of IN A P, the membrane potential remains elevated allowing repetitive bursting (Figure 1-2B) to continue until IN A P 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, IN A P prevents the afterhyperpolarization that follows every spike thereby promoting another burst in the spike train (Figure 1-2A©) (Lee & Heckman, 2001). IN A P is expressed ubiquitously in the PBC; however not all PBC neurons have bursting pacemaker properties even though IN A P appears to perform similar functions in non-pacemaker and pacemaker neurons. Therefore, bursting pacemaker neurons are characterized not just by the presence of IN A P but by a ratio of IN A P to K + leak currents (IK +.L E AK) a n o < by the ability to continue firing when synaptically isolated (Del Negro et al, 2002b; Ptak et al, 2005). IN A P confers bursting properties and increases the excitability of all respiratory neurons (pacemaker and non-pacemaker). However, by interacting with IK+.LEAK. ^ a P may have a more specific role in setting 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, IQ^ (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 VRG (Pena et al, 2004; Del Negro et al, 2005). ICAN is expressed in nearly all PBC neurons; its mechanism is voltage-independent but requires increased cytoplasmic Ca 2 + in order to be activated (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 C A N 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 IN A P and I C A N 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 IN A P or ICAN) and are proposed to compose a kernel of respiratory neurons, 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 IN A P and has been widely used to assess the importance of IN A P 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 5 0 = 2 - 3 p.M), riluzole has been shown to be effective in selectively blocking the IN A P 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 anti-inflammatory drug, blocks the I C A N 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 IN A P and I C A N to respiratory rhythm generation. 1.2. Phytogeny and respiratory rhythm generation 1.2.1. Do phylogenetic differences exist in the roles of I N A P 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 IN A P and ICAN facilitate inspiratory burst generation, they may be important in the recovery from hypothermic respiratory arrest by providing drive to the CRG to initiate breathing. Because adult hamsters are able to recover from respiratory arrest, we hypothesize that hamsters have a greater dependence on IN a P and I C A N 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 IN a P and ICAN are involved with initiating breathing after arrest and therefore asked if the reliance on IN a P 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 A receptor-mediated modulation (Wong-Riley 6k Liu, 2005)(Liu 6k Wong-Riley, 2004). The switch in the expression of GABA A receptor subunits at 12 PND results in a change in the GABA signal (via GABAA) from depolarizing to hyperpolarizing (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 IN a P 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 IN a P 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 N l P and IC A N in respiratory rhythm generation? While most of the current literature examines the role of IN a P and ICAN i n early post-natal 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 IN a P in rhythm generation may be changing as 5 rats mature (Marshall, 2005). The proportion of IN A P 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 GABAA-mediated inhibition in the PBC should counter the role of IN A P and I C A N 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 IN A P and ICAN to maintaining normal respiratory rhythm. We hypothesize that during development changes are occurring in the CRG, particularly in the currents that engender intrinsic bursting (IN A P and ICAN)> that cause the neonate to 'out-grow' the ability to autoresuscitate. We hypothesize that the influence of l N a P and I ^ N on rhythm generation declines so that adult mammals depend less on IN A P and I C A N than neonatal mammals for rhythm generation. To test our hypotheses that phylogenetic and ontogenetic differences occur in the roles of IN A P and ICANI w e eliminated IN A P 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 IN A P 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; A5 , pontine noradrenergic group; BotC, Botzinger Complex; c V R G , caudal ventral respiratory group; IO, inferior olive; KF, Kolliker-Fuse nuclei; LPB, laternal parabrachial nucleus; LRt, lateral reticulum; MPB, medial parabrachial nucleus; MVe, medial vestibular nucleus; Mo5, trigeminal motor nucleus; N A , nucleus ambiguus compactum; NTS, nucleus of the solitary tract; PBC, 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 M ) of a bursting inspiratory cell and rectified, integrated motor output from hypoglossal nerve (XII). Example redrawn from Pace et al. (2007). (B) INaP-mediated pacemaker neuron bursting is represented in black. Grey line predicts action potential trajectory in the absence of I N a P . Please see text for description. 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 Sprague-Dawley rat (Rattus nowegicus; UBC 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 PND. 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 PHR 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 PHR discharge and observable three-phase 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 FFA individually and in combination. When riluzole or FFA 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 FFA 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 FFA 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 FFA 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 2 ) . In these experiments, the preparation was initially perfused and allowed to stabilize with 5% C 0 2 perfusate for 30 - 60 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 2 0). FFA (Sigma, St. Louis, MO) was solubilized with 100 m M N a O H and then titrated to pH 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 2 0 . For all experiments, vehicle 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, UK) . Figure 2-4 shows how the variables measured were derived from the phrenic neurogram (PHR). Inspiratory duration, T h was measured as the time between the onset and end of phrenic bursting; expiratory duration, T E , was measured as the time between the end of a phrenic burst and the onset the following burst. The inverse of the sum of T[ and T E was multiplied by 60 to calculate burst frequency (bursts min'1)- Amplitude of the 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" (nVE). 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 Range Mean ± SD Median (ml/min) (ml/ min) (ml/min) Young rats 8 - 1 8 12.9 ±2.7 12 Weaned rats 3 0 - 4 0 34.4 ± 4.3 32 Weaned hamsters 1 9 - 3 8 29.0 ± 5.3 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 Range (g) Mean ± SD (g) Median (g) Young rats 2 0 - 4 1 28.1 ±4 .0 27.5 Weaned rats 68 - 107 85.7 ± 15.7 82 Weaned hamsters 33 - 100 62.9 ± 18.9 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; Th, thalamus; Pons, pons; SC, superior colliculus. Adapted from Paxinos and Watson (Paxinos & Watson, 1986). 13 . PHR 5 sec B JCVN J 5 sec JCVN C V N JPHR A P H R 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 pre-inspiratory (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 h inspiratory duration; T E , expiratory duration. 15 3. Results In this study, riluzole (IN A P blocker) and flufenamic acid (FFA; lCAN 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 IN A P 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 IN a P 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 INap when applied at low concentrations. At the beginning of 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 (nVE) were quantified, corrected for 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, 10 -12 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, nVE, (Figure 3-2C) in weaned rats did not change significantly from baseline until 18 uM. Young rats significantly decreased nVE at 8 uM, however no significant difference existed between the ages until 12 uM. Hamster preparations decreased nIn-significantly from baseline at low concentrations. To depict the changes in T[ and T E, pyramid plots are shown in Figure 3-3. In a pyramid 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 (TE). To calculate the values for T[ and T E, normalized T[ and T E means corrected for vehicle controls (in 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 E steadily increased with increasing concentrations of riluzole (downward slope shifts right along x-axis). In weaned rats, T E also increased but changed very little. In response to increasing concentrations of riluzole, rat preparations preferentially decreased frequency by elongating the interburst interval. Hamsters (Figure 3-3C) increased both T, and T E (peak and downward slope shift right along x-axis) which resulted in the observed change in frequency. 3.2.Flufenamic acid application Similar to riluzole, FFA 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 FFA starting at 0.25 U.M and increased the concentration of FFA 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 nVE (C), corrected for vehicle controls, in response to increasing concentrations of FFA. Burst frequency (Figure 3-5A) was not significantly affected by the addition of FFA in any of the groups. Amplitude (Figure 3-5B) decreased with increasing FFA 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 uM. Amplitude slowly decreased in young rat preparation and was significantly different from baseline after 10 uM. A significant difference exists between young and weaned rats at low concentrations of FFA. Hamster preparations maintained phrenic burst amplitude near baseline values. 18 Neural ventilation decreased significantly in weaned and young rats, particularly at higher concentrations, but nVE was maintained around baseline in hamsters (Figure 3-5C). Pyramid plots in Figure 3-6 depict the changes in Tj and T E in response to increasing 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 E remained the same with increasing concentrations 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, nVE, 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 IN_P and IQAN- We expected that young rats and hamsters would have a greater decrease in Active breathing resulting from a hypothesized greater dependence on IN a P and ICAN f ° r respiratory rhythm generation. Six concentrations of the riluzole and FFA combination 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% C0 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 nVE, corrected for vehicle controls, of the phrenic 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 nVE occurred in weaned rat preparations where nVE reached <10% of baseline. However, the drop in nVE was due to different components (though the effects are offset so nVE was similarly affected in all groups). In 20 hamster preparations, decreased frequency contributed more to the decrease in nVE; in rat preparations, decreased amplitude contributed to the decrease in nVE. The response of n VE in response to individual application of riluzole and FFA were added to calculate expected nVE values based on the assumption that riluzole and FFA effects are additive. Figure 3-10 compares the expected values with the actual response of nVE. In all groups, 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 nVE was 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, TE increased as concentrations of riluzole and FFA increased. In hamster preparations (Figure 3-11C), T] remained around baseline values and TE increased dramatically up to 37x baseline values at 10 pM riluzole + 8 pM FFA, after which TE values decreased. In response to increases concentrations of riluzole and FFA in combination, rats and hamsters decreased frequency by increasing the interburst interval (TE). 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 z bubbling in the perfusate to 7% (93% 02) and expected that respiratory rhythm would be greater during coapplication of riluzole and FFA than the preparations exposed to 5% CO z. Preparations were exposed to perfusate equilibrated with 7% C 0 2 (93% 02) in order to increase the respiratory drive when the combination of riluzole and 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% CO z , no young rat preparations continued bursting after the final concentration of the cocktail (Table 3-4). The baseline values for preparations perfused with 7% C 0 2 were calculated by dividing the 7% C 0 2 (high drive) value by the 5% C 0 2 (normal drive) value prior to drug application. Table 3-6 shows the frequency, T, and T E baseline (absolute) values for preparations at 5% or 7% C 0 2 . Frequency in rat preparations tended to increase when exposed to 7% C 0 2 (significantly in 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 2 following coapplication of riluzole and FFA. In young rats (Figure 3-12A), frequency was significantly higher at low concentrations for 7% C 0 2 than 5% C 0 2 . This difference disappeared at higher concentrations. No significant difference 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 z maintained amplitude 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 2 and 7% C 0 2 preparations for young rats or hamsters. In all groups, nVE (Figure 3-14) decreased significantly from baseline values in high drive preparations as concentrations of the drug combination increased. In weaned rat preparations (Figure 3-14B), ventilation was increased significantly in 7% C 0 2 . In young rats and hamsters (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 z and coapplication of riluzole and FFA significantly increased ventilation 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 Mean (bursts/min) Range (bursts/min) Young rat 19.4 ± 1.0 6.4-51.5 Weaned rat 18.1+0.9 6.6 - 35.3 Weaned hamster 46.6 ± 4.2 13.4 - 128.6 Table 3-2: The number of animals with phrenic nerve activity at indicated concentrations of riluzole. Nu mber of animals [R1L] Young Weaned Weaned (uM) rats rats hamsters 0.0 11 6 6 0.2 11 6 6 1.0 11 6. 6 2.0 11 6 6 4.0 11 6 6 6.0 11 6 5 8.0 11 6 3 10.0 10 6 2 12.0 5 6 1 14.0 3 6 1 16.0 3 6 0 18.0 3 6 0 20.0 3 6 0 24 Figure 3-1: Thirty second representative raw (PHR) and rectified integrated (JTHR) neurograms of PHR 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 2 0 2 5 [ 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 (nVE, 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 uM RIL 0.2 uM RIL 1 uM RIL 2 uM RIL 4 uM RIL 6 uM RIL 8 uM RIL 10 uM RIL 12 uM RIL 14 uM RIL 16 uM RIL 18 uM RIL 20 uM 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. 2 7 Table 3-3: The number of animals wi ith phrenic nerve activity at indicated concentrations of FFA. Number of animals Concentration Young rats Weaned Weaned (uM) rats hamsters 0.0 10 7 7 0.25 10 7 7 1.25 10 7 7 2.5 10 7 7 5.0 10 7 7 7.5 10 7 7 10.0 9 7 7 12.5 8 7 • 7 15.0 8 7 7 17.5 6 7 7 20.0 6 7 7 22.5 6 7 6 25.0 6 7 6 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 (nVE, 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 1 2 3 4 5 T i m e (sec) Weaned hamsters 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 0.5 1.0 T i m e (sec) 2.0 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. 3 1 0.0 J 1 1 1 1 r— 0 25 50 75 100 [FFA] uM Figure 3-7: Frequency, amplitude and neural ventilation {nVE) all declined as FFA concentration increased in weaned rat preparations (n=6). Open shapes indicate significant difference from 0 uM. Table 3-4: Number of preparations producing respiratory rhythm during coapplication of riluzole and FFA. Number of animals Concentration Young Weaned Weaned [RILMFFA] rats rats hamsters 0 uM+0 u M 16 10 8 6 uM+2.5 u M 16 9 8 7.5 uM+5 u M 14 9 7 9 uM+6 u M 13 9 7 10 uM+8 p M 7 9 6 12pM+10pM 4 10* 6 20pM+15pM 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 JPHR PHR I OuM 7.5uM RIL/5uM FFA 10uM RIL/8u.M FFA 20|iM RIL/15uM FFA u 5 sec |!PHR J U U U U U L j U X L A X l U U l ^ K J u U U J U j ^ ^ j ^ ^ ^ ^ j ^ h . PHR r|i^!;Li.iLi^Lt-iLli-il<, •|.p||iH^..|iii|i»llj|.i'i|i>i^ilj> ^ ^ i ^ ^ ^ . j , >..#.• )i H 5 sec JPHR Jl PHR •y+t# -*,>+Y' V-V""1 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 (nVE, C) corrected for vehicle controls, in response to increasing concentrations of riluzole and FFA. 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 uM (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 (nVE) based on additive action of riluzole and FFA compared to the actual nVE for young rats (A), weaned rats (B) and weaned hamsters (C). Dashed line = baseline =1.0. 35 Young rats 0 nM/O uM 6 u M RIL/2.5 u M F F A 7.5 uM RIL/5 uM 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 2 producing respiratory rhythm during coapplication of riluzole and FFA. Number of animals Concentration Young Weaned Weaned [RILMFFA] rats rats hamsters 0 pM+0 pM 13 6 8 6 uM+2.5 uM 13 6 8 7.5 uM+5 pM 12 6 8 9 pM+6 pM 10 6 8 10 uM+8 pM 7 6 8 12 pM+10 pM 7 6 8 20uM+15 pM 2 6 5 Table 3-6: Alterations in phrenic nerve burst frequency, T ( and T E of young rat, weaned rat and weaned hamster preparations exposed to 5% C O z or 7% C 0 2 . Values are expressed as means±standard error. N=number of preparations. Frequency (bursts/min) T, (sec) T E (sec) 5% 7% 5% 7% 5% 7% Young rat 17.5 ± 1.78 22.6 ± 2.22 0.48 ± 0.02 0.41 ± 0.04 3.67 ±0.67 2.80 ±0.53 N-13 Weaned rat 17.1 ±6.4 21.4 ±3.4 0.82 ± 0.33 0.64 ±0.21 3.41 ±2.58 2.23 ± 0.65 N=6 Hamster 29.4 ± 4.6 29.2 ±3.1 0.57 ± 0.07 0.60 ± 0.08 1.79 ±0.27 1.66 ±0.28 N=8 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 2 or 7% C 0 2 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 2 preparations. 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 z or 7% C 0 2 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 2 preparations. 38 Figure 3-14: Changes in neural ventilation (nVE), corrected for vehicle controls, for young rat (A), weaned rat (B) and hamster (C) preparations exposed to perfusate equilibrated with 5% C 0 2 or 7% C 0 2 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 2 preparations. 39 4. Discussion We hypothesize that phylogenetic and ontogenetic differences occur in the roles of I N a P and ICAN such that hamsters and young rats have a greater reliance on I N a P and ICAN f ° r respiratory rhythm generation than weaned rats. Riluzole (an I N a P blocker) and FFA (an ICAN blocker) were 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 N a P 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 N a P and I C A N for respiratory rhythm generation than weaned rats. These data indicate that a change in the role of I N a P and I C A N in the CRG occurs during development. Increasing excitatory drive to the CRG 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 N a P 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 N a P . [As described in section 1.3, I N a P 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 N a P (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 IN a P. 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 5 0 = 40 pM - 47 pM) (Srinivas 6k Spray, 2003)(Harks et al, 2001), reversibly inhibit Ca2+-dependent and voltage-dependent CI" channels (White 6k Aylwin, 1990), and stimulate or inhibit (depending on concentration) large conductance Ca2+-activated K + channels which may influence neuronal firing (Ottolia 6k Toro, 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 C A N 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 IN a P (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 IN a P and I C A N results in a change in frequency, rhythmogenesis at the CRG would be affected; whereas, a decrease in amplitude would indicate that the elimination of IN a P and IQ^N has decreased the excitability of respiratory network. 4.2. Phylogenetic differences in I N a P 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 INaP to set respiratory rhythm Using the in situ preparation, eliminating IN a P 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 IN a P (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 post-natal 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 N a P 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 (10-20 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 CRG is being affected by the application of riluzole and that in hamsters I N a P plays an important role in neurons that set respiratory rhythm. In contrast with the drastic effect of eliminating IN a P, FFA application had no significant 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 2 +, CI" and K* currents and gap junctions (described in 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 N a P 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 N a P on frequency. Also, at the majority of the concentrations applied (2.5 - 15 pM), FFA would stimulate large conductance Caz+-activated K 4 channels (between 5 - 1 0 pM) (Kochetkov et al, 2000), which may 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 N a P and I ^ N affected the neurons that set respiratory rhythm in the CRG in hamsters, but not in rats. 4.2.2. Respiratory burst amplitude is affected in rats more than hamsters by the elimination of both IN a P and 1^ ^ The elimination of only I N a P 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 N a P is important in promoting repetitive bursting in respiratory neurons (Crill, 1996; Del Negro et al, 2002b). Blocking IN a P, and therefore repetitive bursting, 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 XII 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 XII 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 response1 may have been observed if larger concentrations of 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 (INap) and producing the inspiratory drive potential (ICAN)> eliminating IN A P 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 IN a P and LJAJJ Eliminating I N a P alone in hamster in situ preparations resulted in a significant decrease in neural ventilation (nVE) at lower concentrations than in rat preparations (Figure 3-2C). The decrease in nVF in hamsters was produced by the decrease in both frequency and amplitude; in rats, the decrease in nVE was caused by the decrease in amplitude. That respiratory rhythm generation in hamsters was very sensitive to elimination of I N a P suggests that the hamster respiratory network may be configured differently than the network in rats or that I N a P 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 nVE in rats while nVE in hamster preparations remained around baseline values (Figure 3-5C). In both hamster and rats, any drop in nVE due to FFA was caused by the observed decrease in amplitude (Figure 3-5B). 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 nVE. Applying these drug combinations resulted in significantly decreased nVE in both hamster and rat preparations (Figure 3-9C). We calculated expected values for nVE (Figure 3-10) on the assumption that the effect of the drugs would be additive. Comparing the expected values to the actual nVE values obtained showed that, in the in situ preparation, nVE declined more than was expected if the effects of riluzole and FFA were simply additive. This difference between expected and actual nVE values also highlights the drastic difference between rats and hamsters to the elimination of I N a P and I C A N . More hamster preparations still exhibited fictive breathing following the elimination of I N a P 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 nVE to the same 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 N a P 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 N a P and lCAN reduces the excitability of the respiratory network and removes a source of drive to the CRG thereby decreasing nVE in rat and hamster preparations, increasing the proportion of C 0 2 in the perfusate of the in situ preparation to provide an alternate source of drive to the CRG should counteract the effect of the elimination of I N a P or ICAN-Increasing the proportion of C O z from 5% (Pco2 ~ 42 Torr) to 7% (Pco2 ~ 52 Torr) in the 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 2 tended to be greater, but not significantly, than values at 5% C 0 2 (Figure 3-12C). In intact hamsters exposed to a 5% hypercapnic 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 2 preparations (Figure 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, nVE was not different in 7% C 0 2 preparations than in 5% C 0 2 preparations (Figure 3-14C). In contrast, weaned rat preparations significantly increased nVE (Figure 3-14C) in response to 7% C 0 2 by increasing frequency (Figure 3-12C) and significantly increasing amplitude (Figure 3-13C). These results agree with previous studies in rat in situ preparations which reported that increasing perfusate PCo2 increased phrenic burst frequency (St.-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 2 in the hamster in situ preparations did not change total ventilation or the response to eliminating IN A P and ICAN ; therefore, chemoreceptor drive to the respiratory network was not able to reduce the effects of eliminating l N a P and I C AN - These results suggest that increasing drive cannot replace the effects on rhythm that I N A P and I C A N provide. The estimated P C Q 2 of the 7% C O z is 52 Torr; in vivo 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. We hypothesize that I N a P and ICAN a r e involved with initiating recovery and that reductions in the relative roles of IN A P and I C A N 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 N a P (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 N a P is eliminated by riluzole with E C 5 0 = 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 N a P 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 N a P and ICAN mediate autoresuscitation requires direct testing. 4.3.2. Young rats are more dependent on INaP and ICAN 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 2 \ K + and CI" currents and have the overall affect of reducing excitability of the 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 IN A P through the application of riluzole while the majority of young rat preparations did not (Table 3-3). nVE was decreased more 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 IN A P than weaned rats and may rely more on IN A P for maintenance of rhythm generation. Both rat groups significantly decreased nVE after application of FFA (0.25 - 25 pM) so 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 VE, all declined (Figure 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, nVE decreased significantly in both age groups so that the groups were not significantly different (Figure 3-9C). The drop in n VE was due to the 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 VE existed between the age groups when IN A P and I CA N were eliminated together. 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 2 from 5% to 7% in the perfusate. Amplitude (Figure 3-13A, B) 51 increased in 7% C 0 2 more in weaned rats than in young rats. Therefore, neural ventilation (Figure 3-14A, B) increased in response to 7% C 0 2 to a greater extent in weaned rats than in 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, nVE was greater at 7% C 0 2 than 5% C 0 2 , and it significantly dropped as drug concentrations increased but the significant difference between 7% C 0 2 and 5% C 0 2 preparations was maintained until the final concentration. In contrast, nVE in young rats also increased in 7% C 0 2 preparations but decreased significantly to match the values of 5% C 0 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 2 may to due to 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 2 while the weaned rats have a fully developed hypercapnic ventilatory 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 N a P 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 N a P than in rats. Our results from young rats also suggest that younger rats may be more dependent on I N a P 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 N a P (and ICAN) f ° r rhythm generation may be a neonatal characteristic retained by hamsters into 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 CRG to one that relies more on INaP-mediated bursting neurons (compared to the rat) since expression of ICAN m 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 CRG that is more dependent on INaP-mediated mechanisms of rhythm generation. In addition, in hamsters P C 0 2 is greater and P 0 2 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 N a P 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 IN A P. Hamsters rely more on INaP-mediated mechanisms to set respiratory rhythm than rats. 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 IN A P and IcAN-mediated mechanisms of rhythm generation. Our data are consistent with the hypothesis that a developmental change occurs in the relative roles of IN A P and ICAN in the CRG of mammals. Both hamsters and young rats showed a greater reliance on IN A P and ICAN f ° r rhythm generation than weaned rats; both hamsters and young rats also can autoresuscitate. This correlation is consistent with the suggestion that IN A P and IcAN-mediated bursting neurons may facilitate recovery from hypothermic respiratory arrest. 54 5. References Abdala, APL, Koizumi, H, St. John, W M , Moorgani, B, Smith, JC & Paton, JFR (2004). 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[RIL] (uM) T T E Young rats Weaned rats Hamsters Young rats Weaned rats Hamsters 0.0 1.00 ± 0.00 1.00 ± 0.00 1.00 ±0.00 1.00 ± 0.00 1.00 ± 0.00 1.00 ± 0.00 0.2 1.04 ± 0.02 0.98 ± 0.02 1.12 ±0.06 1.02 ± 0.02 1.07 ± 0.02 1.38 ±0.18 1.0 0.93+0.02 0.96 ± 0.02 1.19 ±0.07 1.07 ± 0.06 1.07 ± 0.04 1.50 ±0.23 2.0 0.88 ± 0.01 0.97 ± 0.04 1.18 ±0.16 1.06 ± 0.08 1.09 ± 0.06 1.89 ±0.31 4.0 0.94 ± 0.03 0.84 ± 0.04 1.15 ±0.08 1.27 ±0.21 1.09 ± 0.08 2.04 ± 0.46 6.0 0.92 ± 0.06 0.83 ± 0.05 1.10 ±0.13 1.75 ±0.40 1.15 ±0.11 2.94 ± 0.45 8.0 0.92 ± 0.08 0.94 ± 0.07 1.29 ±0.17 2.16 ±0.62 1.09 ±0.15 2.98 ± 1.81 10.0 1.02 ±0.17 1.04 ± 0.08 1.05 ±0.22 2.57 ±0.74 1.19 ±0.22 2.66 ± 0.63 12.0 1.11 ±0.31 1.15 ±0.10 1.55 3.31 ± 1.85 1.30 ± 0.27 5.25 14.0 1.46 ±0.67 1.20 ± 0.09 1.42 1.58 ±0.33 1.32 ±0.26 7.15 16.0 1.46 ±0.70 1.21 ±0.14 1.91 ±0.30 1.43 ±0.28 18.0 1.51 ±0.71 1.30 ±0.12 2.84 ± 0.63 1.65 ±0.41 20.0 1.30 ±0.37 1.32 ±0.12 4.48 ± 2.06* 1.72 ±0.32 Table 6-2: T, and T E values for young rats, weaned rats and weaned hamsters in the FFA trials. [FFA] (uM) T, TE Young rats Weaned rats Hamsters Young rats Weaned rats Hamsters 0.00 1.00 ± 0.00 1.00 ±0.00 1.00 ±0.00 1.00 ± 0.00 1.00 ± 0.00 1.00 ± 0.00 0.25 1.10 ±0.16 1.05 ±0.05 0.97 ± 0.03 1.04 ± 0.07 0.99 ±0.11 1.01 ±0.07 1.25 0.98 ± 0.05 1.02 ±0.01 1.07 ± 0.07 0.98 ± 0.07 0.95 ± 0.08 0.97 ± 0.05 2.50 0.87 ± 0.05 0.95 ± 0.03 0.97 ± 0.08 1.00 ± 0.06 0.97 ±0.14 0.91 ± 0.09 5.00 0.81 ±0.07 1.03 ± 0.04 0.89 ±0.12 1.02 ±0.08 0.95+0.12 0.87 ±0.10 7.50 0.81 ±0.06 1.08 ± 0.09 0.87 ±0.11 1.34 ± 0.34 0.92 ±0.10 0.88 ±0.12 10.00 0.70 ± 0.03 1.19 ±0.22 0.62 ± 0.06 0.80 ±0.12 0.99 ± 0.13 0.87 ±0.10 12.50 0.73 ± 0.02 1.24 ±0.29 0.72 ±0.06 0.89 ± 0.20 1.29 ±0.39 0.96 ±0.15 15.00 0.63 ± 0.01 1.24 ±0.33 0.92 ± 0.08 1.23 ± 0.68 1.04 ± 0.09 0.90 ±0.14 17.50 0.61 ±0.03 1.02 ±0.13 0.87 ±0.10 0.52 ± 0.04 1.47 ± 0.42 0.75 ±0.10 20.00 0.64 ± 0.04 1.03 ±0.14 0.88 ± 0.09 0.51 ±0.04 1.11 ±0.11 0.91 ±0.22 22.50 0.64 ± 0.03 0.91 ± 0.09 0.78 ±0.16 0.37 ± 0.03 1.81+0.63 0.76 ±0.18 25.00 0.63 ± 0.04 0.83 ± 0.15 0.66 ±0.16 0.35 ± 0.03 2.12 ± 1.23 1.00 ± 0.32 61 Table 6-3: Ti and T E data for young rats, weaned rats and weaned hamsters in the trials with coapplication of riluzole and FFA. . [ R I L M F F A ] T , T E Young rats Weaned rats Hamsters Young rats Weaned rats Hamsters O u M 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 6 u M / 2 . 5 u M 0.89 ± 0.03 0.84 ± 0.02 1.11 ±0 .12 1.34 ± 0 . 1 1 1.03 ± 0 . 0 8 1.85 ± 0.35 7 . 5 u M / 5 u M 0.92 ± 0.02 0.81 ±0 .02 1.19 ±0 .19 1.54 ± 0 . 1 6 1.11 ±0 .09 3.37 ±0 .75 9 u M / 6 u M 1.04 ±0 .06 0.99 ± 0.06 1.24 ±0 .19 2.14 ± 0 . 3 3 1.32 ±0 .18 15.46 ± 6.32 1 0 U M / 8 U M 1.15 ±0 .09 1.01 ± 0.07 1.26 ±0 .26 2.33 ± 0.38 1.52 ±0 .23 37.42 ±25 .8 1 2 u M / 1 0 u M 1.09 ± 0.09 1.12 ±0 .05 0.99 ± 0.24 2.62 ± 0 . 7 5 2.09 ± 0.56 28.30 ± 17.8 2 0 u M / 1 5 u M 1.30 ± 0 . 3 0 1.33 ±0 .27 3.56 ± 1.02 3.25 ± 0.96 Table 6-4: T[ and T E values for young rats, weaned rats and weaned hamsters in the trials with coapplication of riluzole and FFA [RIL]/ [FFA] T , T E Young rats Weaned rats Hamsters Young rats Weaned rats Hamsters O u M 1.10 ± 0 . 0 8 0.93 ± 0.07 1.01 ±0 .19 1.16 ±0 .07 1.29 ±0 .23 0.94 ±0 .22 6 u M / 2 . 5 u M 1.07 ± 0.09 0.92 ± 0.06 1.12 ±0 .22 1.28 ± 0 . 1 8 1.59 ±0 .47 2.10 ±0 .66 7 . 5 u M / 5 u M 1.28 ±0 .07 1.06 ±0 .10 1.90 ±0 .85 1.95 ± 0 . 5 2 1.72 ±0 .55 4.06 ± 2.24 9 u M / 6 u M 1.17 ± 0 . 0 8 1.11 ±0 .11 3.01 ± 1.62 1.31 ± 0 . 1 3 1.80 ±0 .56 5.64 ± 3 . 3 0 1 0 u M / 8 u M 1.21 ±0 .09 1.10 ±0 .14 5.26 ±2 .76 1.48 ± 0 . 1 8 2.06 ± 0 . 7 1 17.27 ± 14.89 1 2 u M / 1 0 u M 1.25 ± 0 . 1 3 1.21 ±0 .16 4.34 ±2 .16 2.04 ± 0.32 2.17 ±0 .74 6.44 ± 5.03 2 0 u M / 1 5 u M 1.18 ±0 .14 1.13 ±0 .17 1.47 ±0 .39 5.38 ± 4 . 5 6 3.44 ± 1.00 4.19 ± 1 . 3 1 62 

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