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Developmental changes in pontine and adrenergic influences on the respiratory rhythm generator in neonatal… Corcoran, Andrea E. 2005

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D E V E L O P M E N T A L CHANGES IN PONTINE A N D ADRENERGIC INFLUENCES ON THE RESPIRATORY R H Y T H M GENERATOR IN N E O N A T A L RATS by Andrea E. Corcoran B.Sc , Faculty of Science, University of British Columbia, 2003 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF M A S T E R OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES (Zoology) THE UNIVERSITY OF BRITISH C O L U M B I A October 2005 © Andrea E. Corcoran 2005 D E V E L O P M E N T A L CHANGES IN PONTINE A N D ADRENERGIC INFLUENCES ON THE RESPIRATORY R H Y T H M GENERATOR IN N E O N A T A L RATS by Andrea E. Corcoran B.Sc , Faculty of Science, University of British Columbia, 2003 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF M A S T E R OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Zoology) THE UNIVERSITY OF BRITISH C O L U M B I A October 2005 © Andrea E. Corcoran 2005 ABSTRACT In neonatal rats, the pons is known to modulate the respiratory rhythm. Much of the influence is inhibitory and has been attributed to adrenergic inputs originating from the A5 nuclei of the pons. Nonetheless, there are conflicting reports as to how pontine influences change throughout early development in rats. To identify the changes in pontine influence and adrenergic inputs, the in vitro brainstem-spinal cord preparation was used in rats aged PO, P2 and P4 (0, 2 and 4 post-natal days). First, a series of validation experiments was carried out in order to establish the viability and stability of the preparations. PO to P4 preparations were stable (both in Active breathing frequency and burst amplitude) during the first 3 hours of recording. In the second set of experiments, we transected the pons to determine breathing rates with and without pontine inputs. Thirdly, a l and al adrenoreceptor antagonists (Prazosin and Rauwolscine, respectively) were applied to either the pons or medulla, and in the presence or absence of pontine inputs, to determine if results found in the pons transections experiments could be explained by changes in adrenergic inputs. Our pontine transection results showed that at PO, the pons exerted an excitatory drive on the medullary respiratory rhythm generator. At P2, the pons appeared to have no effect on breathing frequency. At P4, the pons switched to providing an inhibition on the rhythm generator. The changes observed with transection of the pons cannot be explained solely by removal of adrenergic influences and must arise from non-adrenergic influences coming from the pons. Blocking a2 adrenoreceptors in the medulla both in the presence and absence of the pons resulted in a decrease in respiratory frequency at all ages. However, medullary al-adrenoreceptor blockade had no significant effect when the pons was still present. Similarly, blocking pontine a2 adrenoreceptors (autoreceptors) did not significantly change breathing frequency. Furthermore, pharmacological blockade of both a l - and a2-medullary adrenoreceptors had a more pronounced effect when the pons was absent, suggesting that the adrenergic inputs are originating not only from the pons, but the medulla itself. Full explanation of the mechanisms underlying the switch from an excitatory pons to one that is inhibitory likely includes changes in several other neuromodulatory systems, and requires further studies using carefully and narrowly defined age groups. ii TABLE OF CONTENTS Abstract i Table of contents ii List of Tables.... '•••v List of Figures vi Acknowledgments x CHAPTER 1. Introduction , 1 1.1 The role of pontine and noradrenergic influences in respiratory control 1 1.1.1 A5 and A6 pontine nuclei 2 1.1.2 Noradrenergic receptor types ; 3 1.1.3 Modulation of the respiratory rhythm generator by A5 and A6 6 A) Medullary effects 6 B) Ponto-medullary effects.. 7 1.2 Developmental changes in pontine influence 8 1.2.1 Scenario A: Pontine inhibition decreases with age in neonatal rats 8 1.2.2 Scenario B: Pontine inhibition increases, then decreases with age in neonatal mice.. 10 1.2.3 Scenario C: Pontine inhibition increases with age 11 1.3 Overall objective 13 CHAPTER 2. Validation experiments 14 2.1 Introduction 14 2.2 Materials and methods 17 2.2.1 Animals ' 17 2.2.2 The neonatal mammal in vitro brainstem spinal cord preparation 18 2.2.3 Experimental protocols 20 A) Stability of the in vitro preparation over time, at different ages 20 B) Brain masses 20 2.2.4 Data analysis 20 2.2.5 Statistical analysis 21 2.3 Results and Discussion .21 2.3.1 Rat: Effects of age on duration and viability of the preparations 21 a) pons ON : 21 b) pons OFF ...23 2.3.2 Rat: Effects of age on frequency over time 23 ii a) Pons ON .• 23 b) Pons OFF 26 2.3.3 Rat: Effects of age on amplitude over time 26 a) pons ON 26 b) pons OFF 29 2.3.4 Rat: Effects of age on burst pattern ...29 a) pons ON -29 b) pons OFF 29 2.3.5 Hamster: Effects of age on duration and viability of the preparations 31 a) pons ON 31 b) pons OFF 31 2.3.6 Hamster: Effects of age on frequency over time 31 a) pons ON 31 b) pons OFF 39 2.3.7 Hamster: Effects of age on amplitude over time 41 a) pons ON .' 41 b) pons OFF ,...,.41 2.3.8 Hamster: Effects of age on burst pattern 44 a) pons ON ...44 b) pons OFF 45 2.4 Discussion 45 CHAPTER 3. Developmental changes in pontine influences on respiratory rhythm generation in neonatal mammals 50 3.1 Introduction ; -50 3.2 Materials and methods 53 3.2.1 Animals and tissue preparation 53 3.2.2 Pharmacological agents.. , . .53 3.2.3 Experimental protocols 54 A) a2-adrenoreceptor antagonist applied to the pons only, in ponto-medullary preparations 54 B) a l and a2-adrenoreceptor antagonists applied to the medulla only, in ponto-medullary preparations : 55 C) a l and a2-adrenoreceptor antagonists applied to the medulla only, in isolated medulla preparations 55 3.2.4 Tissue histology : 56 3.2.5 Data analysis and statistics 57 3.3 Results 57 3.3.1 Histological verification .57 3.3.2 Effects of age and pontine inputs on fictive breathing frequency 58 3.3.3 Effects of age and pharmacological blockade of pontine a2 receptors on fictive breathing frequency 60 3.3.4 Effects of age and pharmacological blockade of medullary a l and a2 receptors on fictive breathing frequency, in the presence of pontine input 61 A) a 1 adrenoreceptor blockade 61 B) a2 adrenoreceptor blockade ". -63 iii .3.3.4 Effects of age and pharmacological blockade of medullary a l and a2 receptors on Active breathing frequency, in the absence of pontine input.. 63 A) a l adrenoreceptor blockade B) al adrenoreceptor blockade 3.4 Discussion 3.4.1 Role of 5-HT1A in modulation the respiratory rhythm: possible confounding factor? , 3.4.2 Sites of catecholamine release A) a l adrenoreceptor effects B) a2 adrenoreceptor effects 3.4.3 A novel scenario for the net developmental changes in pontine influence 3.4.4 Role of other pontine inputs to the medullary respiratory rhythm generator 70 3.4.5 Role of a2 autoreceptors in the pons 73 3.4.6 New models 73 3.5 Conclusions References LIST OF TABLES Table 1.1. Respiratory frequency (bursts/min) recorded from mouse in vitro preparations of various ages with the pons intact or removed (Viemari et al., 2003). At E l 8, most of the preparations were inactive (20/24) while four produced rhythmic bursting (the average of these 4 are presented in the table) 10 Table 1.2. Comparison of two studies showing respiratory burst frequency (bursts/minute) in young and old rat pups with the pons intact or removed. Results from Zimmer (unpublished) are presented in the first two columns, and results from Errchidi (1991) in the last two columns. Note the different definitions of age groups between the two studies 12 Table 2.1. Wet mass of pons, medulla and total (pons + medulla) in milligrams for rats and hamsters aged 0 to 8 days. Data are presented as mean values ± SEM. Note that all rat values are significantly different from hamster values, for each portion of brain weighed of the same age 47 v LIST OF FIGURES Figure 1.1. Dorsal view of a neonatal rat brainstem and cervical spinal cord containing various regions involved in respiratory rhythm generation or modification. Abbreviations: NTS, nucleus of the solitary tract; RTN, retrotrapezoid nucleus; PGi, paragigantocellular reticular nucleus; BotC, Botzinger Complex; preBotC, pre-B6tzinger Complex; pFRG, para-facial respiratory group; rVRG, rostral ventral respiratory group; cVRG, caudal ventral respiratory group; Al /Cl , area of Al/Cl noradrenergic neurons in caudal medulla/rostral spinal cord; A2/C2, area of A2/C2 noradrenergic neursons in caudal medulla/rostral spinal cord; A7, area of A7 noradrenergic neurons in pons; A5, area of A5 noradrenergic neurons in pons; PB, parabrachial nuclei; A6, locus coeruleus; KF, Kolliker-Fuse nucleus. Modified from Rekling and Feldman 1998 3 Figure 1.2. Drawings of adult rat brainstem. The shaded areas represent the location of the individual cell groups at each level. Cell groups A l , A2, A5, A6, and A7 are noradrenergic, and cell groups CI, C2, and C3 are adrenergic. Three levels of CI are indicated. The area of overlap with A l (Al /Cl ) represents the caudal portion, C l m represents the middle portion, and C l r represents the rostral portion. Numbers below plates indicate distance (in mm) caudal to bregma. Abbreviations: 4V, fourth ventricle; cc, central canal; sp5, spinal trigeminal tract; Gr, nucleus gracilis; Sp5C, spinal 5 nucleus, caudal part; NTS, nucleus of the solitary tract; XII, nucleus of cn 12; pyx, decussation of the pyramidal tracts; LRt, lateral reticular nucleus; Ramb, retroambiguous nucleus; AP, area postrema; py, pyramidal tract; ROb, raphe obscurus nucleus; mlf, medial longitudinal fasciculus; 12n, cn 12; Amb, nucleus ambiguus; Sp5I, spinal 5 nucleus, interpolar; MVePC, medial vestibular nucleus, parvicellular; ml, medial lemniscus; LPGi, lateral paragigantocellular nucleus; RVL, rostroventrolateral reticular nucleus; LC, locus coeruleus (A6); SubC, subcoeruleus; spc, superior cerebellar peduncle; mcp, middle cerebellar peduncle; 7n, cn 7; rs, rubrospinal tract; LSO, lateral superior olivary nucleus; RMg, raphe magnus; IC, inferior colliculus; Mo5, motor nucleus of 5; KF, Kolliker-Fuse nucleus; tz, trapezoid body. From Ritter et al., 2001 5 Figure 1.3. Proposed model of the dual A5-A6 modulation of the respiratory rhythm generator in neonatal rodents. Modified from Hilaire et al. 2004 8 Figure 1.4. Occurrence of in vitro phrenic bursts in ponto-medullary and medullary preparations as a function of age. Arrowheads represent transection of the pons from the medulla, (adapted from Viemari et al., 2003) ...11 Figure 2.1. Cross section of the medulla at the level of recording PO2 profiles. Red circles indicate the approximate level of the bilateral PreBotzinger Complexes. Amb, nucleus ambiguous; NTS, nucleus tractus solitarii. Adapted from Okada et al. 1993 15 Figure 2.2. Relationship between age and time at which brains stopped discharging in hamsters and rats with the pons ON or OFF. Data are presented as mean values ± SEM. An asterisk (*) indicates a significant difference between the two preparations joined by the accompanying line Other symbols denote the results of one-way ANOVAs performed between all ages, within a singe type of preparation 22 Figure 2.3. The effect of time on fictive breathing frequency (bursts/min) in rat pons ON (ponto-medullary) preparations of different ages: 0 post-natal days (P0), 2 post-natal days (P2), 4 vi post-natal days (P4), 6 post-natal days (P6), and 8 post-natal days (P8). "First 3 hours" represents the fictive breathing frequncy during the first three hours of recording (or total experiment length for P6 and P8) in preparations of all ages for comparison. Data are presented as mean values ± SEM. Note the differences in time scale in some panels 24 Figure 2.4. Integrated respiratory nerve activity from the first cervical ventral roots of an individual P2 rat pons ON (ponto-medullary) preparation. A) Trace taken 1 hour after start of recording. B) Trace taken 2 hours 32 min. after start of recording : .25 Figure 2.5. The effect of time on fictive breathing frequency (bursts/min) in rat pons OFF (isolated medulla) preparations of different ages: 0 post-natal days (PO), 2 post-natal days (P2), 4 post-natal days (P4), 6 post-natal days (P6), and 8 post-natal days (P8). "First 3 hours" represents the fictive breathing frequency during the first three hours of recording (or total experiment length for P6 and P8) in preparations of all ages for comparison. Data are presented as mean values ± SEM. Note the differences in time scale in some panels 27 Figure 2.6. The effect of time on relative amplitude in rat pons ON (ponto-medullary) preparations of different ages: 0 post-natal days (PO), 2 post-natal days (P2), 4 post-natal days (P4), 6 post-natal days (P6), and 8 post-natal days (P8). "First 3 hours" represents the relative amplitude during the first three hours of recording (or total experiment length for P6 and P8) in preparations of all ages for comparison. Mean values ± SEM for amplitude are normalized as fraction of control values obtained initially at Time = 0 min. Note the differences in time scale in some panels 28 Figure 2.7. The effect of time on relative amplitude in rat pons OFF (isolated medulla) preparations of different ages: 0 post-natal days (PO), 2 post-natal days (P2), 4 post-natal days (P4), 6 post-natal days (P6), and 8 post-natal days (P8). "First 3 hours" represents the relative amplitude during the first three hours of recording (or total experiment length for P6 and P8) in preparations of all ages for comparison. Mean values ± SEM for amplitude are normalized as fraction of control values obtained initially at Time = 0 min. Note the differences in time scale in some panels , 30 Figure 2.8. The effect of time on fictive breathing frequency (bursts/min) in hamster pons ON (ponto-medullary) preparations of different ages: 0 post-natal days (PO), 2 post-natal days (P2), 4 post-natal days (P4), 6 post-natal days (P6), and 8 post-natal days (P8). "First 3 hours" represents the fictive breathing frequency during the first three hours of recording (or total experiment length for P6 and P8) in preparations of all ages for comparison. Data are presented as mean values ± SEM. Note the differences in time scale in some panels .32 Figure 2.9. Integrated respiratory nerve activity from the first cervical ventral roots of an individual P2 hamster pons ON (ponto-medullary) preparation during the entire length of recording (observed at T=27°C). Note the apnea observed during the first hour of recording.. ..33 Figure 2.10. Integrated respiratory nerve activity from the first cervical ventral roots of an individual P2 hamster pons ON (ponto-medullary) preparation during the entire length of recording (observed at T=27°C). Note the irregular breathing pattern throughout the experiment 35 vii Figure 2.11. Integrated respiratory nerve activity from the first cervical ventral roots of an individual P2 hamster pons ON (ponto-medullary) preparation during the first hour of recording (observed at T=27°C). Note the episodic breathing pattern during this recording 38 Figure 2.12. The effect of time on fictive breathing frequency (bursts/min) in hamster pons OFF (isolated medulla) preparations of different ages: 0 post-natal days (PO), 2 post-natal days (P2), 4 post-natal days (P4), 6 post-natal days (P6), and 8 post-natal days (P8); "First 3 hours" represents the fictive breathing frequency during the first three hours of recording (or total experiment length for P6 and P8) in preparations of all ages for comparison. Data are presented as mean values ± SEM. Note the differences in time scale in some panels... /. 40 Figure 2.13. The effect of time on relative amplitude in hamster pons ON (ponto-medullary) preparations of different ages: 0 post-natal days (PO), 2 post-natal days (P2), 4 post-natal days (P4), 6 post-natal days (P6), and 8 post-natal days (P8). "First 3 hours" represents the relative amplitude during the first three hours of recording (or total experiment length for P6 and P8) in preparations of all ages for comparison. Mean values ± SEM for amplitude are normalized as fraction of control values obtained initially at Time = 0 min. Note the differences in time scale in some panels 42 Figure 2.14. The effect of time on relative amplitude in hamster pons OFF (isolated medulla) preparations of different ages: 0 post-natal days (PO), 2 post-natal days (P2), 4 post-natal days (P4), 6 post-natal days (P6), and 8 post-natal days (P8). "First 3 hours" represents the relative amplitude during the first three hours of recording (or total experiment length for P6 and P8) in preparations of all ages for comparison. Mean values ± SEM for amplitude are normalized as fraction of control values obtained initially at Time = 0 min. Note the differences in time scale in some panels.... 43 Figure 2.15. Histograms showing the number of hamster ponto-medullary preparations that displayed an apnea within the first hour of recording (dark shaded bars) or that were breathing continuously (light shaded bars) at various ages (0, 2,4, 6, and 8 post-natal days) 45 Figure 2.16. The effect of the mass of the preparation (both hamster and rat, pons ON and pons OFF) on its duration and viability. A) Values are connected for each preparation type. B) Values are connected for each age group ..48 Figure 3.1 Cross-sections (dorsal side on top) following a DAB reaction of a P4 rat in vitro brainstem spinal-cord preparation that received HRP application on the pons only. HRP stains brown. A: section taken at the level of the pons. B: section taken at the level of the medulla 58 Figure 3.2 Cross-sections (dorsal side on top) following a DAB reaction of a P4 rat in vitro brainstem spinal-cord preparation that received HRP application on the medulla only. HRP stains brown. A: section taken at the level of the pons. B: section taken at the level of the medulla.. ..59 Figure 3.3. Relationship between age and the effect of pontine transection on the fictive breathing frequency of the rat brainstem-spinal cord preparation. Data are presented as mean values ± SEM. An asterisk (*) indicates a significant difference from pons ON control values within the same age group. Bars with the same letter within a treatment group (pons ON or pons OFF) were not significantly different, those with different letters.were. P < 0.05 is considered significant. ...60 viii Figure 3.4. Effect of pharmacological blockade of pontine a-adrenoreceptors with Rauwolscine (Rw) and removal of the pons at different ages in rat en bloc preparations. Data are presented as mean values ± S E M . Values are normalized as fraction of control Values (ie. pons ON). A n asterisk (*) indicates a significant difference from control (ie. pons ON) for each particular age :..61 Figure 3.5. Effect of pharmacological blockade of medullary a-adrenoreceptors and removal of the pons at different ages in rat en bloc preparations. A : medullary a l adrenoceptors were blocked with Prazosin (Pr). B: medullary a2 adrenoreceptors were blocked with Rauwolscine (Rw). Data are presented as mean values ± S E M . Values are normalized as fraction of control values (ie. pons ON). A n asterisk (*) indicates a significant difference from control (ie. pons ON) for each particular age 62 Figure 3.6. Effect of pharmacological blockade of a-adrenoreceptors on rat ponto-medullary and medullary preparations at different ages. Medullary a l adrenoreceptors were blocked with Prazosin (Pr) either in the presence (A) or absence of the pons (B). Medullary a2 adrenoreceptors were blocked with Rauwolscine (Rw) either in the presence (C) or absence of the pons (D). Data are presented as mean values. ± S E M . Values are normalized as fraction of control values (ie. pons O N in A and C, pons OFF in B and D). A n asterisk (*) indicates a difference from control (ie. pons O N in A and C, pons OFF in B and D) for each particular age 64 Figure 3.7. Comparison between our recorded results and those from Errchidi et al. (1991) 69 Figure 3.8. Proposed models 1 and 2 for the medullary adrenergic influence on the respiratory rhythm generator based on results from medullary a2-adrenoreceptor blockade 74 Figure 3.9. Proposed model for the medullary and pontine adrenergic influence on the respiratory rhythm generator at post-natal days 0, 2 and 4 in neonatal rats. This model is based on results from medullary and pontine a l - and a2-adrenoreceptor blockade as well as pons transections. Note that in this figure it is assumed that connections to a 1-adrenoreceptors are originating from A6 while those neurons connecting with a2-adrenoreceptors are originating fromA5 . 75 ix ACKNOWLEDGMENTS First and foremost I would like to thank my supervisor, Bill Milsom. You really are one of a kind and I am so thankful to have been in your lab. You are a constant source of motivation, knowledge and support. You have taught me so many things about research and science, and how to have fun in life. You are a person whom I strive to be, and if I end up even the least bit like you in the future, I will consider myself very lucky. I would also like to thank my committee members, Vanessa Auld and Colin Brauner, for their excellent suggestions and support throughout both the experimental and writing processes, and my departmental examiner Matt Ramer for his constructive feedback. Thank you to everyone in the Milsom lab, both past and present. Each and every one of you has had a positive impact on me and I am very thankful that I was able to share my time in the lab with you. You have made my working environment most importantly fun, but also educational and supportive. I would like to specifically acknowledge Barbara Gajda who performed most of the rat pons ON validation experiments. I would also like to thank some of the Milsom lab neighbours for their help with tissue preparation and histology. Thank you Murry Gilbert for teaching me histological techniques and for your time spent with me using the microscope. Thank you to members of the Ramer lab for your assistance with all things histological. And lastly, thank you to my family and friends who have supported me constantly, and have provided me with a life outside of school. x 1. INTRODUCTION 1.1 THE ROLE OF PONTINE AND NORADRENERGIC INFLUENCES IN RESPIRATORY CONTROL Breathing in mammals is generated by the rhythmic movement of the thoracic cavity, moving air into and out of the lungs, allowing for gas exchange to occur. The respiratory pump muscles causing the movement are innervated by spinal motor neurons, which are in turn controlled by respiratory neurons that generate the primary respiratory rhythm. It has been shown repeatedly that isolated medullary preparations are capable of producing rhythmic respiratory bursts, indicating the existence of a respiratory rhythm generator in the rostral ventrolateral medulla. The pre-B6tzinger complex is currently accepted by most as the kernel for central respiratory rhythm generation (Smith et al., 1991), along with a newly found site known as the para-facial respiratory group (Onimaru and Homma, 2003). Several studies have also reported the presence of neurons with respiratory-related activity in the rostral pons (see Bianchi et al. 1995 for review). These are reported mostly in the medial parabrachial nuclei and the Kolliker-Fuse nucleus, which together form the parabrachial complex (PbC). Respiratory neurons from these areas are involved in the termination of inspiration and therefore in setting motor burst duration, especially when pulmonary feedback is suppressed (see Bianchi et al. 1995; Hilaire et al., 2004 for review). Both glutamate (via NMDA receptors) and GABA-mediated pathways arising from the PbC are involved in this process. Thus, the pons is not necessary for rhythm generation but can influence respiratory rhythm. It is thought that this influence is most likely through the release of endogenous noradrenaline from pontine noradrenergic cells onto the medulla (Hilaire et a l , 1989; Errchidi et al., 1990, 1991). 1 1.1.1 A5 and A6pontine nuclei There are two major pontine groups of noradrenergic cells, the A5 and A6 nuclei (see figures 1.1 and 1.2 for their location). The A6 nucleus is also known as the locus coeruleus and is located in the dorsomedial pons, along the lateral border of the fourth ventricle. It is the largest single source of noradrenergic axons in the central nervous system (Dahlstrom and Fuxe, 1964) and may play a role in initiating or maintaining the stages of the sleep-wake cycle, including the control of the R E M stage of sleep (Foote et al., 1983). A6 is also often associated with chemosensitivity (Oyamada et al., 1998). These neurons discharge at an increased frequency in response to systemic hypercapnia (Elam et al., 1981) and tissue acidosis in the areas surrounding A6 is coupled to a large increase in respiratory frequency (measured in phrenic motor output) (Coates et al., 1993). These results suggest that the intrinsically chemosensitive A6 neurons may function directly as respiratory chemosensors. The A5 group, located in the ventrolateral pons plays a key role in autonomic processes such as cardiovascular regulation and respiration (Dawid-Milner et al., 2001), and casts many projections to cardiorespiratory regions of the brainstem and spinal cord (Fenik et al., 2002). Its involvement in cardiac regulation is somewhat ambiguous, as electrical stimulation of A5 increases blood pressure and heart rate (Loewy et al., 1979), while stimulation of this region with glutamate decreases mean arterial pressure, heart rate and cardiac output (Stanek et al., 1984). The respiratory effects of glutamate injections into A5 of adult rats consist of a decrease in respiratory rate (Dawid-Milner et al., 2001), suggesting that activation of A5 neurons facilitates an expiratory response. Both A5 and A6 receive inputs from the ventrolateral medulla, including adrenergic cells from the CI group and nonaminergic cells from surrounding areas (Guyenet et al., 1993). These projections are of interest as the ventrolateral medulla is an area that plays a major role in respiratory rhythm generation. 2 Figure 1.1. Dorsal view of a neonatal rat brainstem and cervical spinal cord containing various regions involved in respiratory rhythm generation or modification. Abbreviations: NTS, nucleus of the solitary tract; R T N , retrotrapezoid nucleus; PGi , paragigantocellular reticular nucleus; BotC, Botzinger Complex; preBotC, pre-B6tzinger Complex; pFRG, para-facial respiratory group; r V R G , rostral ventral respiratory group; c V R G , caudal ventral respiratory group; A l / C l , area of A l / C l noradrenergic neurons in caudal medulla/rostral spinal cord; A2/C2, area of A2/C2 noradrenergic neursons in caudal medulla/rostral spinal cord; A 7 , area of A7 noradrenergic neurons in pons; A5 , area of A5 noradrenergic neurons in pons; PB, parabrachial nuclei; A6 , locus coeruleus; K F , Kolliker-Fuse nucleus. Modified from Rekling and Feldman 1998. 1.1.2 Noradrenergic receptor types Neurons in A5 and A6 communicate with other cells by releasing noradrenaline (NA) which binds mainly to alpha noradrenergic receptors (or a adrenoreceptors) on the post-synaptic cell. There are two types of alpha adrenoreceptors: a l and a2 (Langer, 1974; Berthelsen and Pettinger, 1977). Alpha-1 receptors are entirely post-synaptic and are coupled to phospholipase C and phosphoinositol turnover (Nicoll et al., 1990). Activation of a l adrenoreceptors results in the excitatory action of NA. Most commonly, activation of a l adrenoreceptors decreases resting 3 potassium conductance thus strongly depolarizing sensitive neurons (Finlayson et al., 1986). Conversely, activation of a2 adrenoreceptors results in an inhibitory action by hyperpolarizing neurons (Nicoll et al., 1990). These neurons are hyperpolarized by an increase in potassium conductance resulting from the activation of a2 adrenoreceptors (Williams et al., 1985). Alpha-2 adrenoreceptors are coupled to G-proteins that are negatively coupled to adenylate cyclase and can either block calcium channels or open potassium channels. Unlike the a l receptors, a2 receptors are located both pre- and post-synaptically (Nicoll et al., 1990). Adding complexity to the network of noradrenergic source neurons arid their targets is the presence of autoreceptors on A5 and A6 cells. Alpha-2 adrenoreceptors are present on the cell surface of both A5 and A6 neonatal rat neurons (Huangfu et al., 1997). In both cases, a2 adrenoreceptor activation results in an increase of inwardly rectifying potassium conductance, such that if these cells are stimulated with NA, they will turn off (by hyperpolarization) or be functionally inhibited. The presence of these receptors on noradrenergic cells may allow them to autoregulate their own activity, or the activity of neighbouring noradrenergic cells by collateral inhibition. Additionally, a transient population of a l adrenoreceptors on locus coeruleus neurons grown in culture has been found, and this transient population may explain age-related differences in response to NA (Finlayson et al., 1986). In cells over 26 days old, N A stimulation resulted in a hyperpolarization, mediated by inhibitory a2 adrenoreceptors. However, cells less than 26 days old exhibited a biphasic hyperpolarization and depolarization mediated by both a2 and a l adrenoreceptor activation respectively. Consistent with this, light microscopic autoradiographic localization of adrenergic receptors revealed a high density of a2 receptors and a low density of a l receptors in young A6 cells (Young and Kuhar, 1980). 4 Figure 1.2. Drawings of adult rat brainstem. The shaded areas represent the location of the individual cell groups at each level. Cell groups A l , A 2 , A 5 , A6 , and A7 are noradrenergic, and cell groups C I , C2, and C3 are adrenergic. Three levels of C I are indicated. The area of overlap with A l ( A l / C l ) represents the caudal portion, C l m represents the middle portion, and C1 r represents the rostral portion. Numbers below plates indicate distance (in mm) caudal to bregma. Abbreviations: 4V, fourth ventricle; cc, central canal; sp5, spinal trigeminal tract; Gr, nucleus gracilis; Sp5C, spinal 5 nucleus, caudal part; NTS, nucleus of the solitary tract; XII, nucleus of cn 12; pyx, decussation of the pyramidal tracts; LRt, lateral reticular nucleus; Ramb, retroambiguous nucleus; AP, area postrema; py, pyramidal tract; ROb, raphe obscurus nucleus; mlf, medial longitudinal fasciculus; 12n, cn 12; Amb, nucleus ambiguus; Sp5I, spinal 5 nucleus, interpolar; MVePC, medial vestibular nucleus, parvicellular; ml, medial lemniscus; LPGi, lateral paragigantocellular nucleus; RVL, rostroventrolateral reticular nucleus; LC, locus coeruleus (A6); SubC, subcoeruleus; spc, superior cerebellar peduncle; mcp, middle cerebellar peduncle; 7n, cn 7; rs, rubrospinal tract; LSO, lateral superior olivary nucleus; RMg, raphe magnus; IC, inferior colliculus; Mo5, motor nucleus of 5; KF , Kolliker-Fuse nucleus; tz, trapezoid body. From Ritter et al., 2001. 5 1.1.3 Modulation of the respiratory rhythm generator by A 5 and A6 A) Medullary effects The medullary respiratory rhythm generator is subject to a tonic noradrenergic inhibitory drive originating from the A5 noradrenergic nucleus in the pons, and a tonic noradrenergic excitatory drive originating from the A6 nucleus (Hilaire et al., 1989; Errchidi et al., 1990). Application of exogenous noradrenaline (NA) to medullary preparations of neonatal rats resulted in a decreased breathing frequency (Hilaire et al., 1989; Errchidi et al., 1990, 1991). The NA depressive effect was mediated through medullary a2-adrenoreceptors, as this respiratory response to N A was blocked by pre-treatment with a2-receptor antagonists (Hilaire et a l , 1989; Errchidi et al., 1990, 1991). The al-receptor antagonist, Prazosin, did not block the NA-induced decrease in respiratory frequency; rather it potentiated the rhythm depression (Errchidi et al., 1991). This latter finding suggests that there are medullary al-adrenoreceptors that contribute to excitation of respiratory rhythm. Thus, when endogenous noradrenaline is released onto the medulla, it elicits both a l excitatory and a2 inhibitory effects. In rats, a2 inhibition masks the weak a l excitation. The opposite is found in mice, where exogenous NA applied to only the medulla increases breathing frequency - an effect mediated predominantly by the a l excitatory adrenoreceptors which mask a weak inhibition from a2 adrenoreceptors (Viemari and Hilaire, 2002). Yet despite the predominance of a l excitatory receptors within the mouse medulla, mouse pontomedullary preparations remain inactive, suggesting that pontine inhibition dominates the medullary respiratory rhythm (Viemari et al., 2003). The net excitatory effect of exogenous NA on the mouse medulla suggests there may be a strong influence of descending input from A6. Involvement of A6 neurons in respiratory rhythm modulation was further explored in a recent study by Hilaire and colleagues (2004). Using mice with genetically-induced alterations of their noradrenergic neurons, they were able to provide evidence that A6 neurons exert a facilatory effect on rhythm generation via medullary a l -6 adrenoceptors. Proper development is required of both A5 and A6 neurons for a normal respiratory rhythm at birth. In Rnx mutants, where formation of A5 neurons is drastically impaired, respiratory frequency is abnormally high at birth (Shirasawa et al., 2000). While in Phox2a mutants, where A6 neuronal development is compromised, respiratory frequency is abnormally low in fetuses delivered at E l 8 (Viemari et al., 2004). The same effect is seen in Ret mutants who possess fewer A6 neurons (Hilaire et al., 2004). B) Ponto-medullary effects N A applied to pontomedullary preparations of neonatal rats resulted in an increased breathing frequency, while N A applied to preparations without the pons induced a decreased frequency. Errchidi and colleagues (1991) suggested that the increase was due to an inhibition of A5 neurons by N A (implication of autoreceptors). Thus, they suggested that N A applied to the medulla mimics the endogenous effects of N A on A5 medullary targets. While the firing of A5 neurons is inhibited by activation of al autoreceptors when N A is applied to the pons (Huangfu et al., 1997), resulting in a disinbition of A5 influence on the medulla. In summary, it is postulated that there exists an opposite modulation of rhythm generation from the A5 and A6 pontine groups (see Figure 1.3). A5 neurons project and release noradrenaline onto inhibitory al adrenoreceptors in the medulla while A6 neurons project to the excitatory a l adrenoreceptors. Both types of neurons appear to be present at birth and are required for proper development of the respiratory rhythm generators although the net balance of the two receptor types varies across species and developmental age. Finally, autoreceptors are now known to be present on A5 and A6 neurons but their role in modulating breathing as a function of species and age is unknown. 7 Respiratory frequency modulation Figure 1.3. Proposed model of the dual A 5 - A 6 modulation of the respiratory rhythm generator in neonatal rodents. Modified from Hilaire et al. 2004. 1.2 DEVELOPMENTAL CHANGES IN PONTINE INFLUENCE In the literature, there is general agreement that the pons provides an inhibitory input onto the medulla during the neonatal stage, and that this inhibition attenuates into adulthood. Whether or not the inhibition is only present transiently during the early stages of development, or whether it persists throughout life remains a debate. Also in dispute is the pattern through which pontine inhibition changes throughout the neonatal period. The following scenarios depict different observations of the changes in pontine inhibition made by various research groups. 1.2.1 Scenario A: Pontine inhibition decreases with age in neonatal rats In early studies, Errchidi et al. (1991) found that in rats, A5 inhibition was stronger just at birth than after a few post-natal days. In preparations with the pons still intact, frequency increased from post-natal day 0 to 4 (ie. P0-P4). If the pons was removed, frequency did not change with increasing age. However, more recent studies have shown that A5 inhibition of the respiratory rhythm generator persists through adulthood (Jodkowski et al., 1997). 8 Yet more recently, further arguments have been made not only for the attenuation of pontine inhibitory influences, but also for a possible increase in excitatory drive. Morphological changes in both A5 and A6 sites have now been reported as development proceeds (Ito et al., 2002). The number of A5 neurons decreases as age increases (Pl-10), while the volume of the A6 area does not change. Thus the ratio of A5:A6 neurons decreases. This suggests that pontine inhibition decreases with age due to a net postnatal reduction in the number of A5 neurons. An alternative (or possibly complementary) theory is that excitatory effects of A6 neurons increase with age (despite no change in volume), thus counteracting inhibition from the A5 region. Thus, in a recent study by Hakuno (2004), where A6 neurons were inactivated via microinjections of Tetrodotoxin (TTX) or N A (TTX blocks fast sodium channels, thus inhibiting the generation of action potentials while NA activates a2-adrenergic receptors on the somatodendritic membrane of A6 neurons, eliciting a hyperpolarizing (ie. inhibitory) response (Williams et al., 1985)), a decrease in respiratory frequency was observed in P3-4 rats but no change was observed in PI-2 rats. Also, electrical stimulation of A6 resulted in an increase in frequency in P3-4 preparations whereas no change was observed in the younger group. Combined, these results suggest that A6 exerts an excitatory effect on the medullary respiratory rhythm generator in an age-dependent manner. Along with the previous findings from Ito (2002) and Errchidi (1991), it is proposed that there is a shift during early development in the relative roles of the "inhibitory" A5 area and the "excitatory" A6 area. : Finally, while the study of Hakuno (2004) suggests an increasing role of the excitatory A6 area during development, Errchidi (1991) showed that NA applied to the medulla over this time period caused a decrease in frequency. This has led to the suggestion that noradrenergic output from A6 does not directly project onto the PreBotC, but may project onto, and use A5 as a relay station. Thus, A6 could release NA onto A5 a2 autoreceptors, thus inhibiting A5 inhibitory 9 output. Electrical stimulation of A6, however, did not elicit any significant change in neuronal activity in the A5 area (Hakuno et al., 2004). 1.2.2 Scenario B: Pontine inhibition increases, then decreases with age in neonatal mice Much of the above evidence of pontine influence has been obtained from studies using neonatal rats. Slight differences are observed in studies of mouse maturation where the role of the pons passes through several transitional stages (Viemari et al., 2003). It appears that the pons has no effect on breathing frequency in early embryonic stages; a similar pattern of spontaneous bursts is observed both in embryological day 16 (ie. El6) preparations with the pons on or with the pons removed (see Table 1.1 and Fig. 1.4). Inhibitory modulation emerges at approximately El8 where some preparations discharge with the pons on and some do not. Following this stage, the pons becomes so inhibitory that ponto-medullary preparations of E l 8 to P5 rats remain completely inactive. Pontine inhibition then weakens and between P6-P9, respiratory bursts are generated at a low frequency with the pons on and increase when the pons is removed. Note that preparations of P6-P9 were short-lasting. The authors attributed this to poor brainstem oxygenation when the age and size of the preparation increased. Compared to the rat, pontine inhibition is significantly stronger in the mouse. Table 1.1. Respiratory frequency (bursts/min) recorded from mouse in vitro preparations of various ages with the pons intact or removed (Viemari et al, 2003). At E l 8, most of the preparations were inactive (20/24) while four produced rhythmic bursting (the average of these 4 are presented in the table). E16 E18 P0-2 P3-5 P6-9 Pons ON 6.6±0.7 4-5 (in 4/24 preps) 0 0 3.3±0.5 Pons OFF 4.6±0.9 10.9±0.6 11.1 ±0.8 8.9±0.5 8.5±1.2 10 E 16 E 18a E 18b JILL! P0-P2 juimiL P3-P5 P6-P9 LLJJUJ Lujy 20 s Figure 1.4. Occurrence of in vitro phrenic bursts in ponto-medullary and medullary preparations as a function of age. Arrowheads represent transection of the pons from the medulla, (adapted from Viemari et al., 2003) 1.2.3 Scenario C: Pontine inhibition increases with age In contrast to the results described in section 1.2.1, where pontine inhibition appears to decrease with age in rats, Zirnmer (unpublished) suggested that pontine inhibition increases with age as removal of the pons significantly increased frequency in older preparations only (see Table 1.2). Discrepancies between this study and that of Errchidi et al. (1991) may be due to the difference in age group definitions: "young" was 0-1 days vs. 0-2 days, and "old" was 2-4 vs. 4-6 days in the Errchidi et al. and Zimmer studies respectively. Another possibility may lie in the level of transections made (not detailed in either study). However, Zimmer's findings parallel some of the observations made in mouse in vitro preparations (described in section 1.2.2). In the 11 mouse preparations, there was a transition between pontine input that was mildly inhibitory at E16-E18, to input that was so inhibitory that it abolished breathing in preparations aged E18-P5. Since Zimmer did not go past P6, it is difficult to predict if a decrease in inhibition would be observed in rats older than this, as seen in the mouse preparations. Table 1.2. Comparison of two studies showing respiratory burst frequency (bursts/minute) in young and old rat pups with the pons intact or removed. Results from Zimmer (unpublished) are presented in the first two columns, and results from Errchidi (1991) in the last two columns. Note the different definitions of age groups between the two studies. A. Data from Zimmer B. Data from Errchidi (1991) Y o u n g O l d Y o u n g O l d ( P O - 2 ) ( P 4 - 6 ) ( P O - 1 ) ( P 2 - 4 ) P o n s O N 8 . 6 6 . 7 4 3 . 8 5 . 5 P o n s O F F 9 . 7 6 1 2 . 4 3 9 . 2 1 0 . 0 Finally, other experiments have shown a relationship between the presence of the pons, age and degree of cold tolerance of neonatal rat brains. In older (P4-6) rat neonate preparations when the pons was removed, fictive breathing continued at lower temperatures than in preparations with the pons intact (21.9°C pons on, 16.9°C pons off) (Zimmer and Milsom, unpublished). It was suggested that because the pons exerts more of an inhibitory effect on rhythm generation in older animals, removing the pons and its inhibitory input on motor output allowed for further cooling before respiratory arrest occured. In young (PO-2) preparations, the presence of the pons had no effect on the temperature of arrest (19.7°C pons on, 20.2°C pons off), consistent with the suggestion that it also had no tonic inhibitory influence. 12 1.3 O V E R A L L OBJECTIVE Given the discrepancies that exist in the literature, the objective of this study was to further investigate developmental changes in pontine excitatory and inhibitory influences in neonatal rats. Firstly, to identify which scenario of changes in pontine influence occurs in our hands, we recorded fictive breathing frequency in preparations (0, 2, and 4 post-natal days) before and after transection of the pons. By using pharmacological agents that block a l and a2 adrenoreceptors in the medulla and the pons, we hoped to distinguish between the relative roles of A5 and A6 throughout early development and to detect changes in adrenoreceptor distribution with age. It was hypothesized that at times when the pons was more inhibitory, blocking a2 adrenoreceptors in the medulla would have a larger effect on breathing frequency than at times when the pons was less inhibitory. It was also hypothesized that blocking a l adrenoreceptors in the medulla would have less effect on breathing frequency when the net influence of the pons was inhibitory. Also, to tease out the role of pontine a2 adrenoreceptors (most likely A5 and A6 autoreceptors), a2 adrenoreceptor antagonists were applied to the pons only. It was hypothesized that blocking these receptors would disinhibit A5 and A6 cells, and further increase their drive. Before beginning the pharmacological experiments, it was first necessary to conduct a series of validation experiments. These experiments were designed to determine the stability and viability of rat brainstem spinal cord preparations and different ages (both with the pons intact and removed). Similar experiments were performed on hamsters as a comparison since preliminary data suggest that hamsters behave in a fashion similar to mice (Zimmer and Milsom, 2004). 13 2. VALIDATION STUDY 2.1 INTRODUCTION Al l experiments were performed in the following study using the in vitro brainstem-spinal cord preparation, otherwise known as the en bloc preparation. The en bloc preparation was first adapted for use in neonatal mammals by Suzue (1984) and has repeatedly been used for studies involving respiratory rhythm generation in neonatal rodents ever since. Briefly, it consists of an isolated brainstem and spinal cord that is constantly superfused with artificial cerebrospinal fluid gassed with 95%02/5%C02 to keep the tissue alive. Recordings can be made from nerves that innervate respiratory muscles in the intact animal. However, the physiological viability of the preparation has been questioned because, as it lacks blood perfusion, all respiratory gases and metabolites must diffuse from the superfusate through the tissue. Depth profiles of PO2 in the isolated brainstem-spinal cord of neonatal rats were first made by Okada et al. (1993). Although they did not specify differences in ages (0 to 5 post-natal days were used), they stated that P02 of the medulla reached 0 at a depth of 450 um. Since it is thought that the central rhythm-generating network is located starting at 200-300 um from the ventral side, it is likely that its environment is hypoxic (Rekling and Feldman,,1998). In contrast, the lowest PO2 measurement taken from the medulla of a working heart brainstem preparation (where the preparation is perfused artificially through the descending aorta) was 29 Torr, and the average was 293.7 ± 45 Torr (Wilson et al., 2001). Also, as can be seen in Figure 2.1, the outer layer of the medulla is hyperoxic. Clearly, various areas in the en bloc preparation are suffering from everything from hyperoxic O2 toxicity to anoxia when compared to the more "normal" in situ state and this must be kept in mind when using this preparation. 14 ventral dorsa\ ^ m b ' a m b ' f i u u s nucleus N T S : nucleus of the solitary tract F i g u r e 2.1. Cross section of the medulla at the level of recording P 0 2 profiles. Red circles indicate the approximate level of the bilateral PreBotzinger Complexes. Amb, nucleus ambiguous; NTS, nucleus tractus solitarii. Adapted from Okada et al. 1993. The underlying objective of this set of validation experiments was to investigate whether results arising from studies of differences associated with pontine influences and age were not simply a result of an increase in oxygenation to the tissue when the pons was removed or a decrease in oxygenation when the preparation was larger as the animal grew. For example, the large increase in bursting frequency seen in older preparations when the pons was removed may have been due to removal of a significant amount of tissue that effectively blocked oxygen diffusion. There was no increase in bursting frequency in younger preparations when the pons was removed (Zimmer and Milsom, unpublished), suggesting perhaps that at this stage in development, the brainstem and spinal cord were small enough to allow adequate tissue oxygenation even with the pons present. Also, most researchers cannot record from a brainstem-spinal cord preparation in animals older than P6 (with larger brains), and if they do, the preparation is extremely short-lasting as nerve output becomes extremely weak further supporting this possibility (see Ballanyi et al., 1999 for review, and Richter et al. 2001 for slice work in older neonates). These next experiments describe the breathing pattern of four different preparations: neonatal rat and hamster preparations with the pons intact (ponto-medullary preparation) or 15 removed (isolated medulla preparation). Previous attempts have failed to obtain a signal in hamster pons on preparations (Zimmer and Milsom, 2004), similar to what is observed in mouse preparations where an intact pons completely disables rhythmogenesis (Viemari and Hilaire, 2002). In an earlier study on mice by Jacquin et al. (1999), spontaneous activity could be seen in the brainstem-spinal cord preparation with the pons still intact only when the perfusate was bubbled with 8% C 0 2 , and not when bubbled with the standard 5% C 0 2 . From this it was suggested that pontine structures tonically depress respiration and that high C O 2 levels overcome this depressive pontine influence. Also, in the isolated brainstem-spinal cord of neonatal hamsters with the pons intact, although no breathing was observed at 23°C, spontaneuous fictive breathing appeared when the preparation was being warmed to 27°C (Zimmer and Milsom, 2004). However, this breathing was short-lasting and disappeared once the temperature stabilized at 27°C. These observations suggest that activity can be recorded from a hamster preparation with the pons intact under some circumstances. The first aim of these experiments was to quantify the stability of en bloc preparations of different ages and the duration over which these preparations generate activity under control conditions. By looking at the activity of the preparation over time, we were able to determine how long it remains stable and at which point it deviates from initial recordings, thus suggesting the maximum duration of future experiments. These experiments served as a control allowing us to determine if changes seen in later trials were due to experimental manipulations or to inherent differences in the motor output over time (eg. deterioration of signal or pattern alterations). As previously mentioned, many researchers suggest that older preparations (P6 and above) do not last as long due to a decreased level of tissue oxygenation (resulting from increased size). In my experiments, size was always an issue as rat brains are generally larger than hamster brains for any given age, and ponto-medullary preparations are larger than isolated medulla preparations. Thus, a second objective for the validation studies was to shed some light 16 on the hypothesis that larger preparations "die" sooner due to a lack in tissue oxygenation (which is less of a factor for the younger, smaller brains, or brains with the pons removed). The four different types of preparations were appropriately chosen to allow for a comparison to be made between different ages, while taking into account the size of each preparation. By comparing the size of the brain with the length of time the preparation was able to discharge, we hoped to gain some insight into the oxygenation levels of the various preparations. Rat pup brains are on average twice the size of hamster pups of comparable ages. Therefore, if it is true that the rat preparations fail to discharge at older ages due to size (most experiments in the literature are performed on rats), then hamster pup preparations should work at older ages than rats, since their brains will not reach the size of a rat brain of similar age until at least several days later. Also, ponto-medullary preparations are larger than isolated medulla preparations and it may therefore be argued that the former preparations are receiving less oxygen and therefore may not last as long. Thus, quantification of preparation viability coupled with weights of all preparations (both species, both types of preparations, all ages) should provide data that either support or refute the hypothesis that lack of oxygen due to increased diffusion distance is the reason that older preparations (especially with the pons on) are less viable. 2.2 METHODS 2.2.1 Animals Two species were used in these experiments, Sprague-Dawley rats and Syrian hamsters. Pregnant Syrian hamsters were purchased from Charles River Rodent Laboratories (Stone Ridge, NY, USA) and allowed to give birth naturally. Hamster pups were randomly separated from their mother immediately prior to an experiment. The rat pups were obtained from the UBC Animal Care Centre the morning of an experiment and kept warm under a heating lamp until used. Both 17 species of pups were divided into 5 age groups: PO (day of birth), P2 (2 post-natal days), P4 (4 post-natal days), P6 (6 post-natal days) and P8 (8 post-natal days). 2.2.2 The neonatal mammal in vitro brainstem spinal cord preparation First, the animal was placed in a chamber and anesthetized with 2-4% halothane. The animal was then removed and the skin cut along the dorsal midline from the top of the skull to the abdomen. A quick decerebration was performed by cutting bilaterally from the midline along bregma and removing tbe face and cerebrum. Any remaining cerebrum was removed with forceps. The thorax was cut off by sectioning the body just rostral to the white milk line. The front limbs and skin were removed and the remaining body transferred to a dissecting dish containing room temperature (~23°C) artificial cerebral spinal fluid (aCSF composed of (in mM) 113.0 sodium chloride, 3.0 potassium chloride, 1.2 sodium phosphate, 1.5 calcium chloride, 1.0 magnesium chloride, 30.0 sodium bicarbonate and 30.0 dextrose) equilibrated with 95% oxygen and 5% carbon dioxide (pH 7.4). Remaining tissue and muscle were removed from the neck region. The animal was placed on its ventral side and two pins were placed in the thorax to fix the preparation to the base of the dish. The skull was split along the midline, opened up and pinned laterally. The telencephalon and colliculi were transected. The spinal cord was exposed by making two lateral cuts through the vertebrae starting from the rostral end. The bone flap and top of the vertebral column were removed. The cord was severed at T12 and the tip held with forceps while dorsal and ventral roots were cut as distally as possible, in the rostral direction. Cranial nerves were cut and the brainstem and spinal cord were transferred to a 2mL recording dish (made of plexiglass with a stainless steel grid separating the chamber into an upper and lower compartment) supplied with a constant flow of ~27°C oxygenated aCSF (at a rate of 5ml/min). Under a dissection microscope, the preparation was cut 1) at the most rostral level of the pons (for a ponto-medullary preparation), or 2) at the pontomedullary junction, just rostral to the anterior, inferior cerebellar artery (for an isolated medulla preparation) using fine opthalmic scissors. The dura was removed and the spinal cord transected at the level of the 7 t h cervical root. The brainstem-spinal cord preparation was then pinned ventral side up. The aCSF was regulated by passing it through a temperature controlled bath (Lauda Model RC6) and then supplied to both the upper and lower compartments of the recording chamber. The temperature of the aCSF perfusate was kept at 27°C, because at this temperature the amplitude of the responses are largest and best maintained (Suzue, 1984). Also, it has been observed that at higher temperatures, the preparation tends to deteriorate quickly (Suzue 1984; personal observation), probably due to increasing oxygen and glucose demands. Temperature was recorded by placing a thermocouple probe into the bathing fluid. A tightly fitting glass suction electrode was attached to one of the first cervical ventral rootlets (CI) to measure respiratory related burst discharge. Although CI is not usually associated with respiratory-related motor discharge, it is in these preparations (Zimmer and Milsom, 2004) and generally provided a strong, robust signal. The phrenic nerve rootlet (C4) discharge has previously been measured simultaneously and compared to CI activity to ensure that CI discharge does in fact correspond to respiratory activity (Smith et al., 1990). The CI nerve rootlet innervates the infrahyoid muscles in the neck, some of which help to depress the hyoid bone after elevation during speech and mastication, and may also function to decrease the inward suction of soft tissue during inspiration. It has been suggested that this may be important in neonatal breathing movements as the hard cartilage and bones are not completely developed and thus the system is flexible and compliant (Hilaire and Duron, 1999). The electrical signals recorded from the glass suction electrode were amplified, filtered and recorded (2000 samples/sec) on Windaq computer data acquisition software (DI200; DataQ instruments, Akron, OH, USA). 19 2.2.3 Experimental Protocols A) Stability of the in vitro preparation over time, at different ages Animals were dissected as described above. Recording (via suction electrode on spinal nerve CI) began as quickly as possible. The average time for dissection and set-up of the preparation until recording began was 10 minutes. Two types of preparation were used for each species (ponto-medullary and isolated medulla), each with N=6 for almost every age. It was difficult to obtain a signal for P8 isolated medulla preparations of either species. Consequently, the Ns for these two groups are low (N-3 for hamsters, N=2 for rats). Fictive breathing was recorded at a sample rate of 2000 samples/sec until there appeared to be no more activity in the preparation, at which point the experiment was terminated. B) Brain masses Brains were weighed from both rats and hamsters at ages PO, P2, P4, P6 and P8. Mass was used as an indication of size. Brains from the ponto-medullary preparations of the previous experiment were used, as well as other brains that were dissected out of new animals. The pons was then transected at a level just rostral to the anterior inferior cerebellar artery and weighed. Following this the medulla and spinal cord were weighed collectively. A total brainstem mass was obtained by adding the pons and medulla/spinal cord weights together. 2.2.4 Data Analysis Samples taken for analysis of fictive breathing frequency and amplitude differed slightly depending on the type and age of preparation used. Efforts were made to sample as consistently as possible, but some changes were made to accommodate the short time older preparations lasted and variability of the hamster ponto-medullary preparations. For rat ponto-medullary preparations and isolated medulla preparations from rats and hamsters at PO, P2 and P4, 2 minute samples were taken every half hour, beginning immediately upon recording (ie. time 0). These 20 samples were taken until the preparation stopped discharging/For older animals, 2 minute samples were taken at more frequent intervals. For the younger (PO, P2 and P4) hamster ponto-medullary preparations, the fictive breathing pattern was extremely variable. Thus, samples were averaged over every half hour. The older (P6 and P8) preparations were more stable and 2 minute samples were taken at intervals similar to those for the three other types of preparations. The raw nerve recordings were full wave rectified and integrated. Fictive breathing frequency was averaged for all individuals in any given type of preparation, at a given time point, and are reported as bursts/minute. Due to the variability in signal strength with each preparation, peak amplitudes were normalized to the time 0 value (control) for each animal and are reported as a value relative to control. 2.2.5 Statistical Analysis To examine the effect of age on the length of time that a preparation was able to discharge, a one-way analysis of variance (ANOVA) on ranks was performed (as normality was not met in any group) followed by a Tukey or Dunn's post hoc test. P < 0.05 was considered significant. The same methods were used to determine whether there was a significant difference between the preparation types at each age. A student's t-test was used to determine significant differences between hamster and rat brain masses (pons, medulla and total masses). 2.3 RESULTS 2.3.1 Rat: Effects of age on duration and viability of the preparations a) Pons ON As can been seen in Figure 2.2, the viability of the rat pons ON preparation decreased with age. Preparations from PO and P2 rats were equally viable, running on average for 354.9±37.1 minutes (~6 hours) and 333.8±64.8 minutes respectively. By P4, the length of time that fictive breathing could be recorded dropped by more than half to 135.8±12.9 minutes. 21 F i g u r e 2.2. Relationship between age and time at which brains stopped discharging in hamsters and rats with the pons ON or OFF. Data are presented as mean values ± SEM. An asterisk (*) indicates a significant difference between the two preparations joined by the accompanying line. Other symbols denote the results of one-way ANOVAs performed between all ages, within a singe type of preparation. 22 Preparations from P6 and P8 rats were short lasting; fictive breathing was recorded for less than an hour (30.3±7.0 and 15.7±4.1 minutes respectively). Due to small sample sizes (n=6 for each age), the differences between most groups were not statistically significant. However, caution must be used when taking these statistical results into account as non-parametric tests were used because the data were not normally distributed. b) Pons OFF The viability for rat pons OFF preparations varied slightly when compared to the rat pons ON preparations. Although viability decreased overall with age, the viability peaked at P2, where preparations ran on average for 844.7±127.6 minutes or 14.1±2.1 hours (see Figure 2.2). This is double the length of time of rat pons ON preparations of similar age. P2 pons OFF preparations lasted significantly longer than P4, P6 and P8 pons OFF preparations. However, the longest recording made was from a PO animal, which went for 23 hours and 17 minutes. There was a dramatic decrease in the time until burst discharge stopped from P6 to P8 animals. P6 preparations lasted on average 69.1±34.8minutes, while P8 preparations bursted for only 3 minutes. Note that this length of time is much shorter than that of the larger rat pons ON preparations of similar age, which lasted almost 16 minutes. Also, it was difficult to obtain a signal from P8 preparations. Out of nine attempted recordings (each from a different preparation), only two were successful. 2.3.2 Rat: Effects of age on frequency over time a) Pons ON Overall, fictive breathing frequency appeared to be maintained over time, regardless of age until just before the preparations failed (see Figure 2.3). An exception to this trend was seen in PO 23 60 120 180 240 300 360 420 480 540 600 Time (min) 60 120 180 240 '300 360 420 480 540 600 Time (min) 60 80 100 120 Time (min) Time (min) 4 6 8 10 Time (min) 0 20 40 60 80 100 120 140 160 180 200 Time (min) Figure 2.3. The effect of time on fictive breathing frequency (bursts/min) in rat pons ON (ponto-medullary) preparations of different ages: 0 post-natal days (P0), 2 post-natal days (P2), 4 post-natal days (P4), 6 post-natal days (P6), and 8 post-natal days (P8). "First 3 hours" represents the fictive breathing frequncy during the first three hours of recording (or total experiment length for P6 and P8) in preparations of all ages for comparison. Data are presented as mean values ± SEM. Note the differences in time scale in some panels. 24 preparations where frequency was maintained around 11 bursts/min for the first hour and half before it started to decline linearly to 0. Initial recordings at time 0 were often slightly higher than those taken half an hour later. On average, frequency for P2 preparations was 9.7±1.2 bursts/min initially and then fell to 7.6±1.7 bursts/min within half an hour. Initial frequency for P4 preparations was 10.9±1.0 bursts/min. It declined to 6.7±0.8 in half an hour and was maintained there. PO and P6 values did not differ much between time 0 and 30 minute recordings, and P8 animals maintained a frequency around 9.5 bursts/min for the entire length of recording (only 16 minutes). Values for P2 were extremely variable, resulting in large error bars when averaged. This most likely reflects changes in pattern and burst shape seen in most of these preparations. There was a tendency for breathing to become episodic, followed by a fractionation in burst discharge (see Figure 2.4). This made it difficult to distinguish between an episode of several breaths, and one breath that was fractionated. A B 1 s e c Figure 2.4. Integrated respiratory nerve activity from the first cervical ventral roots of an individual P2 rat pons ON (ponto-medullary) preparation. A) Trace taken 1 hour after start of recording. B) Trace taken 2 hours 32 min. after start of recording. 25 b) Pons OFF Starting values varied slightly as a function of age (see Figure 2.5). Immediately upon recording, the average fictive breathing frequency for PO was 8.7±1.8 bursts/min. It decreased to 7.1±1.3 bursts/min within a half hour where it remained for three and a half hours before starting to decline. The initial fictive breathing frequency for P2 rats was 11.2±0.9 bursts/min, however it stabilized at 9.3±0.8 bursts/min after half an hour. It began to decline at hour 3. The initial fictive breathing frequency of P4 preparations was 9.0±1.5 bursts/min and it remained at this level for the first hour and a half. P6 preparations exhibited a frequency of 9.0±0.5 bursts/min and P8 9.8±0.5 bursts/min at time 0. Note that most preparations stabilized at around 9 bursts/min over the first half hour except the PO preparations which were lower, breathing at around 7 bursts/min. Patterns of activity towards the end of the experiment (ie. When the preparations stopped discharging) in the older preparations differed from those observed in the younger preparations. In P6 and P8 preparations, fictive breathing frequency remained constant over time (see Figure 2.5). In P0-P4 preparations, however, fictive breathing frequency decreased linearly with time, and the rhythm became irregular towards the end of the experiment. However, these differences in frequency appear to reflect the longevity of the preparations. For example, if we superimpose the fictive breathing frequency of the first three hours (or total experiment time for P6 and P8 preparations, which did not last three hours) for each age group, it can be seen that frequency remained roughly constant over this time span for all groups (Figure 2.5). It is only when the preparations last beyond these first few hours that a decrease in frequency becomes apparent. 2.3.3 Rat: Effects of age on amplitude over time a) Pons ON Amplitude was maintained for almost the entire length of experiments for PO, P2 and P4 preparations, before declining sharply towards the end of recordings (see Figure 2.6). The trend 26 Figure 2.5. The effect of time on fictive breathing frequency (bursts/min) in rat pons OFF (isolated medulla) preparations of different ages: 0 post-natal days (PO), 2 post-natal days (P2), 4 post-natal days (P4), 6 post-natal days (P6), and 8 post-natal days (P8). "First 3 hours" represents the fictive breathing frequency during the first three hours of recording (or total experiment length for P6 and P8) in preparations of all ages for comparison. Data are presented as mean values ± SEM. Note the differences in time scale in some panels. 27 4> ? S = c Q. O E £ re o <o c > 2 5 ^ n 1 1 1 1 1 r 0 60 120 180 240 300 360 420 480 540 600 Time (min) 40 60 80 Time (min) i r 100 120 140 HO.O 160 4 6 8 10 12 Time (min) -|o.o 16 60 120 180 240 300 360 420 480 540 600 Time (min) P6 f i l l 1 J [ 1 10 20 30 40 50 Time (min) 60 70 —•— PO First 3 hours —O— P2 - • — P4 —5— P6 —•— P8 o - o ^ o 20 40 60 80 100 120 140 160 180 200 Time (min) Figure 2.6. The effect of time on relative amplitude in rat pons ON (ponto-medullary) preparations of different ages: 0 post-natal days (P0), 2 post-natal days (P2), 4 post-natal days (P4), 6 post-natal days (P6), and 8 post-natal days (P8). "First 3 hours" represents the relative amplitude during the first three hours of recording (or total experiment length for P6 and P8) in preparations of all ages for comparison. Mean values ± S E M for amplitude are normalized as fraction of control values obtained initially at Time = 0 min. Note the differences in time scale in some panels. 28 was different for the older preparations (P6 and P8) where amplitude began decreasing immediately upon recording and continued to decrease until indistinguishable from background noise levels. This difference in trends is evident in Figure 2.6 in the panel superimposing data for the first three hours for all age groups. Note that none of the values fall to 0. This is because there was a continuous level of baseline noise at roughly 0.3 the control amplitude. Therefore any burst that was 0.3 or lower than control values was not detectable, b) Pons OFF The way in which amplitude changed over time in rat isolated medulla preparations was slightly different than that of the rat ponto-medullary preparations (see Figure 2.7). The amplitude in pons OFF preparations eventually decreased over time in all age groups. It was maintained for the first 3.5 to 4 hours in PO and P2 preparations, but declined after that. In older preparations (P4, P6 and P8), amplitude began to decrease immediately upon recording. 2.3.4 Rat: Effects of age on burst pattern a) Pons ON In the younger rat ponto-medullary preparations (PO and P2) an episodic breathing pattern was evident in almost all recordings. These often developed during the first hour of recording. This pattern often transformed into a series of bursts that were very fractionated (see Figure 2.4). The older preparations (P4, P6, and P8) displayed a more continuous breathing pattern that did not become episodic. b) Pons OFF The pattern in these preparations was fairly consistent throughout all experiments. Bursting was usually continuous and regular, with no apneas or episodes. 29 0» — . 3 £ £ C Q. O (6 O 0) C > 2 11 IT «T ^  ^ «* Time (min) ^ <f> l £ o£ r$ t$ c£ o£ r?> rK> l& G?> Time (min) r e m o > .2 ^ ^ ^ •F&'&'&'jr-Time (min) Time (min) Time (min) Time (min) Figure 2.7. The effect of time on relative amplitude in rat pons OFF (isolated medulla) preparations of different ages: 0 post-natal days (PO), 2 post-natal days (P2), 4 post-natal days (P4), 6 post-natal days (P6), and 8 post-natal days (P8). "First 3 hours" represents the relative amplitude during the first three hours of recording (or total experiment length for P6 and P8) in preparations of all ages for comparison. Mean values ± S E M for amplitude are normalized as fraction of control values obtained initially at Time = 0 min. Note the differences in time scale in some panels. 30 2.3.5 Hamster: Effects of age on duration and viability of the preparations a) Pons ON In contrast to previous attempts, we were successful in obtaining fictive breathing from hamster ponto-medullary preparations (possible explanations for this are considered in section 2.3.8). The hamster ponto-medullary preparations were as viable as the rat isolated medulla preparations (see Figure 2.2). There was a tendency for viability (the time until burst discharge stopped) to decrease as age increased. However, a peak was again seen at P2, where preparations lasted on average the longest (756.0±111.7 minutes or 12.6±1.9 hours). The P8 preparations were the shortest-lived, averaging 21.9±4.4 minutes in length, although this was the maximal average duration for all types of P8 preparations (both rats and hamsters, pons ON and OFF). b) Pons OFF Again, there was a trend for decreasing viability with increasing age beyond P4, with a peak duration at P2 (854.4±117.7 minutes or 14.2±2.0 hours) (see Figure 2.2). The P4 preparations lasted significantly longer than other types of preparation of similar age. They continued to discharge for 530.4±74.0 minutes or 8.8±1.2 hours, which is comparable to PO preparations of the same type (7.8±2.1 hours). It was difficult to obtain a signal from P8 preparations, as was the case with the rat pons OFF preparations. Out of five attempted recordings, only three were successful. Those that were successful persisted on average 16.2±8.2 minutes. 2.3.6 Hamster: Effects of age on frequency overtime a) Pons ON Breathing frequency appeared to be roughly maintained in preparations of all ages (see Figure 2.8). In PO preparations, however, frequency was extremely variable, as is evident by the large error bars. This most likely results from the unusual frequency oscillations, apneas and episodic breathing often observed in these preparations (see Figures 2.9, 2.10 and 2.11). Breathing 31 ~i 1 1 1 1 1 1 r 0 120 240 360 480 600 720 840 960 1080 0 120 240 360 480 600 720 840 960 1080 Time (min) Time (min) 0 100 . 200 300 400 500 600 Time (min) 10 20 30 40 50 60 Time (min) 10 20' 30 Time (min) 40 ' 60 80 100 120 140 160 180 200 Time (min) F i g u r e 2.8. The effect of time on fictive breathing frequency (bursts/min) in hams te r pons O N (ponto-medullary) preparations of different ages: 0 post-natal days (P0), 2 post-natal days (P2), 4 post-natal days (P4), 6 post-natal days (P6), and 8 post-natal days (P8). "First 3 hours" represents the fictive breathing frequency during the first three hours of recording (or total experiment length for P6 and P8) in preparations of all ages for comparison. Data are presented as mean values ± SEM. Note the differences in time scale in some panels. 32 10 min Figure 2.9. Integrated respiratory nerve activity from the first cervical ventral roots of an individual P2 hamster pons O N (ponto-medullary) preparation during the entire length of recording (observed at T=27°C). Note the apnea observed during the first hour of recording. 33 Hour 5 Hour 7 10 min F i g u r e 2.9. (con t 'd ) Integrated respiratory nerve activity from the first cervical ventral roots of an individual P2 hamster pons O N (ponto-medullary) preparation during the entire length of recording (observed at T=27°C). Note the apnea observed during the first hour of recording. 34 Hour 1 10 min Figure 2.10. Integrated respiratory nerve activity from the first cervical ventral roots of an individual P2 hamster pons ON (ponto-medullary) preparation during the entire length of recording (observed at T=27°C). Note the irregular breathing pattern throughout the experiment. 35 Hour 5 mm Hour 6 MM Hour 7 Hour 8 10 min Figure 2.10. (cont'd) Integrated respiratory nerve activity from the first cervical ventral roots of an individual P2 hamster pons ON (ponto-medullary) preparation during the entire length of recording (observed at T=27°C). Note the irregular breathing pattern throughout the experiment. 36 Hour 9 11 ill i 1 1 Hour 10 Hour 11 M . J i l . i l i l L l j Hour 12 10 min Figure 2.10. (cont'd) Integrated respiratory nerve activity from the first cervical ventral roots of an individual P2 hamster pons O N (ponto-medullary) preparation during the entire length of recording (observed at T=27°C). Note the irregular breathing pattern throughout the experiment. 37 10 min Figure 2.11. Integrated respiratory nerve activity from the first cervical ventral roots of an individual P2 hamster pons O N (ponto-medullary) preparation during the first hour of recording (observed at T=27°C). Note the episodic breathing pattern during this recording. 38 frequency in P2 preparations was much more stable, as well as much lower than PO preparations. The average value at the start of P2 experiments was 1.7±0.4 bursts/min, which increased to a maximum of 2.5±0.8 bursts/min after half an hour. In contrast, PO values taken at comparable times were 4.9±1.5 bursts/min and 5.5±1.9 bursts/min. The average starting frequency for P4 preparations was 5.0±1.0 bursts/min, which dropped dramatically to 2.0±0.4 bursts/min after the first 20 minutes, before starting to increase slightly over the following hour and a half before declining again. By P6 and P8, breathing frequency appeared to be a little more steady and consistent. Most breathing frequency values for P6 preparations remained at around 5 bursts/min during the length of the experiment, while average P8 breathing frequency hovered around 6 bursts/min. b) Pons OFF For hamster pons OFF preparations, the pattern of fictive breathing frequency over time varied slightly, depending on the age of the animal (see Figure 2.12). PO and P2 preparations displayed a similar trend, although starting and peak values were drastically different. The initial frequency for PO preparations was 12.9±1.2 bursts/min and it increased steadily over the first few hours until it peaked at 19.5±2,2 bursts/min after 2.5 hours. Note that this frequency is much higher than values taken at similar times in hamster pons ON preparations. Frequency was maintained at this level for another hour before starting to decline linearly for the remainder of the experiment. P2 preparations initially discharged at 8.1±1.3 bursts/min, a value only 2/3 that of the PO preparations at a similar time point. These preparations also increased in frequency over the first 2 hours and peaked at 13.4±1.9 bursts/min (similar value to starting frequency of PO preparations). Fictive breathing frequency began to decline slowly at this point, and after around 5 hours decreased more sharply. 39 Time (min) Time (min) * # <P & 24 • P4 24 -22 - 22 ->» 20 • 20 -o e 18 • 18 -<u 3 C .16 - 16 -req i/mi 14 • 14 -row 12 • i 12 • .E 3 10 - \ - ^ • 10 -'—' 3 . 8 -n a> k. 6 • I T 6 -CD 4 • 4 -2 2 -0 - 0 -lP (S> g? cf> # i> (S> tS> c?> # Time (min) 10 20 30 40 Time (min) 10 15 20 25 30 35 0 20 40 60 80 100 120 140 160 180 200 Time (min) Time (min) Figure 2.12. The effect of time on fictive breathing frequency (bursts/min) in hamster pons OFF (isolated medulla) preparations of different ages: 0 post-natal days (P0), 2 post-natal days (P2), 4 post-natal days (P4), 6 post-natal days (P6), and 8 post-natal days (P8). "First 3 hours" represents the fictive breathing frequency during the first three hours of recording (or total experiment length for P6 and P8) in preparations of all ages for comparison. Data are presented as mean values ± SEM. Note the differences in time scale in some panels. 4 0 P4 preparations also decreased in frequency with time. However, these preparations did not show an increase in the first hour, as was seen with PO and P2 preparations. The initial frequency for P4 preparations was 11.1±0.9 bursts/min. Frequency dropped to around 9 bursts/min within half an hour and was maintained there for 2.5 hours before continuing to decline linearly. The older preparations (P6 and P8) maintained frequency throughout the experiments. P6 preparations started at 7.5±0.9 bursts/min and did not change much over the time course of the experiments. Also, variability in the P6 fictive breathing frequency was low. Values for P8 preparations were not as consistent, although it must be kept in mind that only a small sample size was obtained (n=3). The initial frequency for P8 preparations was 5.8±0.9. Note that values for fictive breathing frequency were much lower in the older preparations than the younger ones. 2.3.7 Hamster: Effects of age on amplitude over time a) Pons ON Amplitude in PO and P2 preparations was maintained for roughly the first five hours before it began to decline linearly for the remainder of the experiment (see Figure 2.13). P4 preparations appeared to show an initial increase in amplitude. Relative amplitude increased to a maximum of 1.56±0.28 the initial value after the first hour of recording. At this point, the average amplitude began to decrease but with a lot of variability. Amplitude of P6 and P8 preparations showed a similar pattern over time. Both decreased consistently and immediately upon recording. b) Pons OFF Data for relative amplitude of hamster pons OFF preparations was much more variable than values for fictive breathing frequency (see Figure 2.14). Again, there seemed to be an increase in amplitude early in the recording for PO and P2 animals. After half an hour from initial recording, amplitude increased to 1.45±0.17 times the initial value for PO preparations. Within an hour it began to decrease steadily. Although there appeared to be a second increase in the figure, this 41 0 120 240 360 480 600 720 840 960 Time (min) 120 240 360 480 600 720 840 960 1080 Time (min) Time (min) 10 20 30 40 Time (min) 60 Time (min) 0 20 40 60 80 100 120 140 160 180 200 Time (min) Figure 2.13. The effect of time on relative amplitude in hamster pons ON (ponto-medullary) preparations of different ages: 0 post-natal days (P0), 2 post-natal days (P2), 4 post-natal days (P4), 6 post-natal days (P6), and 8 post-natal days (P8). "First 3 hours" represents the relative amplitude during the first three hours of recording (or total experiment length for P6 and P8) in preparations of all ages for comparison. Mean values ± S E M for amplitude are normalized as fraction of control values obtained initially at Time = 0 min. Note the differences in time scale in some panels. 42 2.0 i 1.8 -1.6 -pn rol 1.4 -mpli o o 1.2 -ns o 1.0 -w c > o 0.8 -lati "G CD 0.6 -Re l_ H— 0.4 -0.2 -0.0 -Time (min) (S oO oO c© <fi »0 c£ o£ c$ r)& Time (min) •T # # # ^ # ^ ^ ^ Time (min) 10 20 30 40 Time (min) 60 First 3 hours 10 15 20 25 Time (min) 20 40 60 80 100 120 140 160 180 200 Time (min) F i g u r e 2.14. The effect of time on relative amplitude in hams te r pons O F F (isolated medulla) preparations of different ages: 0 post-natal days (P0), 2 post-natal days (P2), 4 post-natal days (P4), 6 post-natal days (P6), and 8 post-natal days (P8). "First 3 hours" represents the relative amplitude during the first three hours of recording (or total experiment length for P6 and P8) in preparations of all ages for comparison. Mean values ± S E M for amplitude are normalized as fraction of control values obtained initially at Time = 0 min. Note the differences in time scale in some panels. 43 was only due to the sample size getting smaller (as preparations were dying out). P2 preparations showed a more pronounced increase in amplitude. Relative amplitude increased steadily until it peaked at 1.64±0.19 after three hours. At this point it decreased progressively. Conversely, P4 preparations did not show this initial increase, but maintained initial amplitude for about 3.5 hours before starting to decrease. Again we saw an apparent increase in amplitude as time went on, but this was due to the sample size decreasing. P6 and P8 preparations showed the same consistent pattern of decreasing amplitude throughout the entire experiment. 2.3.8 Hamster: Effects of age on burst pattern a) Pons ON Current results offer possible explanations for the inability of previous studies to record a signal from a pons on hamster preparation. In many cases of our study, a dozen bursts (or more) can be seen upon initial recording. However, these bursts soon disappear only to return 30 minutes to an hour later, creating a long apnea during the first hour of recording. Thus, if investigators failed to start recording within 10 minutes following initial dissection, the early bursting activity may have been missed. The frequency of this behaviour changed with development (see Figure 2.15). Two thirds of PO preparations showed an apnea during the first hour, whereas all P2 preparations displayed this partem. The incidence of long apneas decreased after this age. Only one P6 preparation out of six showed an apnea and none of the P8 preparations did. The length of all apneas was variable and no correlations could be made with age. Also, as stated earlier, frequency in this type of preparation was extremely variable. This was in part due to the prevalence of an episodic breathing pattern, as well as a pattern that was very irregular (see Figures 2.9, 2.10 and 2.11). 44 apnea within 1st hour ! continuous from start 2 4 A g e Group (days post-natal) Figure 2.15. Histograms showing the number of hamster ponto-medullary preparations that displayed an apnea within the first hour of recording (dark shaded bars) or that were breathing continuously (light shaded bars) at various ages (0, 2, 4, 6, and 8 post-natal days). b) Pons OFF Pattern in preparations with the pons intact differ from those with the pons removed. Like the rat pons OFF preparations, hamster pons OFF preparations were much more stable and displayed a continuous breathing pattern instead of one that was episodic. 2.4 DISCUSSION Based on results from these validation experiments, it can be concluded that most preparations (excluding hamster pons ON preparations) are stable and usable for varying periods up to P4. At these ages, preparations remain robust and last long enough for a several hour experiment to be performed. On average, frequency and amplitude remain constant in hamster and rat pons OFF preparations from PO to P4 for four hours before starting to deteriorate. This suggests that an experiment could be performed with a maximum duration of 4 hours. The rat pons ON preparations begin to deteriorate earlier and thus experiments with this preparation 45 should be limited to a maximum of 2 hours. Also of interest is the smaller variability in rat pons OFF fictive breathing frequencies when compared to the rat pons ON values. The standard errors around rat pons OFF mean values are much smaller, even though both preparations had similar sample sizes (n=6 for each age, except P8). This suggests that pons OFF preparations are more stable and consistent. Hamster pons ON preparations were so unpredictable and inconsistent (in terms of overall breathing pattern and frequency) that one could not confidently depend on this preparation for quatitative experiments. It would be difficult to conclude whether effects seen were due to experimental manipulation or to inherent variability in the preparation. Also, the duration and viability of P6 preparations was extremely variable (20-52 minutes in pons OFF hamsters, 33 minutes to 3h50min in pons OFF rats). It is very difficult to record a signal from a P8 pons OFF preparation (2 out of 9 in rats, 3 out of 5 in hamsters), and if a signal was attained, the duration of the preparation was extremely short-lasting and variable (4.5 to 32 minutes in hamsters, ~3 minutes in rats). This suggests that older preparations cannot be relied on to last very long. The data also suggest that preparations should be allowed to stabilize for a minimum of 30 minutes before baseline values are recorded. Initial values taken immediately upon recording at time 0 often differ (are faster) from samples taken half an hour later, when most seem to have stabilized. Initial recordings may be uncharacteristically high due to excitation caused by the transection of the pons immediately prior to recording. Patterns of activity towards the end of the experiment (ie. when the preparation stops discharging) in the older preparations differ from those observed in the younger preparations. In P6 and P8 preparations, breathing frequency remained constant while burst amplitude decreased linearly with time. In P0-P4 preparations, the rhythm became irregular towards the end of the experiment. This suggests that the deterioration of preparations at different ages is due to different mechanisms. Since pattern is affected over time in the younger preparations, it is 46 possible that the cessation of bursting in these preparations may be due to a failure at the central rhythm-generating network. Because motor output also decreased, however, there must be other mechanisms involved (such as neuronal death in the motor nerve itself). In older preparations, since frequency was not affected and the way in which most preparations stopped discharging was through a decrease in amplitude, failure at the central rhythm-generating network seems unlikely and failure appears to occur at some downstream site. As can be seen in Table 2.1, rat brains were consistently larger than hamster brains at any given age. However, when comparing the viability data (ie. time until burst discharge stopped) between rats and hamsters and between pons ON and pons OFF as a function of mass, values were remarkably similar (see Figure 2.2). This does not support the arguments made by many researchers that older brains are not viable because they are larger which decreases oxygen diffusion, resulting in less neuronal discharge. Note that an ideal measurement, rather than brain mass, would be the distance from the brain surface to the Pre-Botzinger complex (which is the distance that oxygen is required to diffuse to reach the rhythm generating cells). However, this is technically difficult and thus in our study, brain mass was used as a proxy. Table 2.1. Wet mass of pons, medulla and total (pons + medulla) in milligrams for rats and hamsters aged 0 to 8 days. Data are presented as mean values ± SEM. Note that all rat values are significantly different from hamster values, for each portion of brain weighed of the same age. BRAIN M A S S (mg) A G E : 0 2 4 6 8 Hamster Rat Hamster Rat Hamster Rat Hamster Rat Hamster Rat P o n s Medul la Total 10.6±0.7 26.0±1.2 18.5±0.9 30.1+0.9 29.1+0.9 56.2+2.0 16.7±9.0 35.8±1.7 24.5+1.4 37.1 ±0.7 41.2+1.8 72.8+1.8 21.0±1.1 46.4+1.6 33.9+.1.9 48.9+1.9 54.9+1.7 95.2+2.7 27.1±3.9 45.5±1.5 39.7+1.9 55.6±0.8 65.1+3.0 101.3+1.8 33.8+2.5 49.9±1.1 52.0+4.9 67.1±1.3 84.3±2.2 116.3+1.9 47 I 1 1 1 1 1 1 1 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 Mass of preparation (g) i i 1 1 1 1 1 1 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 Mass of preparation (g) F i g u r e 2.16. The effect of the mass of the preparation (both hamster and rat, pons O N and pons OFF) on its duration and viability. A ) Values are connected for each preparation type. B ) Values are connected for each age group. 48 If we look at the direct effect of brain mass on the viability of each type of preparation, several statements can be made. It appears that in early development, age is more of a factor than the weight of the brain in determining the length of time that the preparation will continue to discharge. When we compare the time of death with brain weight, younger preparations lasted longer for any given weight with the exception that PO brains did not last as long as P2 brains (Fig. 2.16). However, this may hold true only until a threshold size is reached, after which the brain size is more of a factor. For example, all brains less than 40-45 mg ran for roughly the same amount of time (-14 hours) while preparations that were larger than 40-45 mg (the size of P2 hamster pons ON preparations) seemed to stop discharging more quickly than preparations that weighed less. Note that for larger brains, pons ON preparations lasted longer than pons OFF preparations (the pons ON preparations here are younger and have a smaller medulla than the older pons OFF preparations). Thus, my data suggest that the viability of the en bloc preparation is affected by developmental changes initially, as well as by size once they surpass a mass of roughly 40mg. 49 3. DEVELEOPMENTAL CHANGES IN PONTINE AND NORADRENERGIC INFLUENCES IN NEONATAL RATS 3.1 I N T R O D U C T I O N In the literature, there are conflicting reports of developmental changes in pontine inputs to medullary respiratory rhythm generators as previously outlined in Chapter 1. The present series of experiments attempted to resolve this conflict by using narrowly defined age groups to more accurately resolve the developmental changes that are occurring in neonatal rats. The experiments were run on three different age groups of neonatal rat (PO, 2 and 4). These ages were used as in vitro preparations from rat pups of these ages were determined to be consistently viable for the time required to run a complete experiment (as justified in the validation experiments, Chapter 2), and spanned the ages over which different results have been reported by others (Zimmer (unpublished) and Errchidi (1991) both recorded from P0-P4 rat pups). In the first set of experiments, Rauwolscine (an a2-adrenoreceptor antagonist) was dissolved in the aCSF bathing only the pons to enhance adrenergic influences from the pons via blockade of a2 adrenoreceptors, particularly autoreceptors on A5 and A6 cells. This will block autoinhibition at each site as well as reciprocal inhibition between sites. Both A5 and A6 would now be unfettered and we should see the net balance of the full expression of each group of cells. Interpreting specific effects could be difficult (if not impossible) however, because it will be influenced by the amount of autoinhibition at each site as well as the amount of reciprocal inhibition at each site. However, if A5 was more dominant, we would expect to see a decrease in breathing frequency because more endogenous NA would be released from A5 neurons onto a2 adrenoreceptors in the medulla than from A6 onto a l adrenoreceptors under these conditions. If the pons was then removed, breathing frequency should increase. If, on the other hand, A6 was 50 more dominant, then when a2 adrenoreceptors on A6 neurons were blocked by Rauwolscine (an a2 adrenoreceptor antagonist), we would expect to see an increase in breathing frequency because the excitatory effects of A6 would be increased. When the pons was then removed, a decrease in frequency would be expected as the excitatory predominant influence of endogenous NA was removed. It was hypothesized that application of Rauwolscine and removal of the pons should exert opposite effects early in development but, due to the conflicting reports in the literature it was uncertain whether this would also occur with increasing age. To further clarify the mechanisms by which the effects of pontine input change in early development, both a l and a2 antagonists were applied only to the medulla, in the presence of pontine input. As mentioned in Chapter 1, there have been three scenarios proposed to describe recorded changes in pontine effects during the early stages of life in rodents. Scenario A describes a decrease in pontine inhibition with age. Scenario B (based on mice) describes an increase in inhibition, followed by a decrease in inhibition with age. Scenario C describes an increase in pontine inhibition with age. By removing the pons and recording breathing frequency at ages PO, P2 and P4, as well as by pharmacologically blocking medullary adrenoreceptors, we hoped to be able to resolve which of these scenarios was correct. One explanation for the reported developmental changes seen in pontine influence on fictive breathing frequency lies in changes in the abundance of a-adrenoreceptors in the medulla. Based on previous findings that A5 cells project to a2-receptors and A6 cells project to a l -receptors (as discussed in section 1.1.3), based on the changes in pontine input that occurred in Scenario C (preliminary experiments and previous work from our lab support this scenario), we put forward the following hypothesis. We hypothesized that there are more a l receptors at PO, whose stimulation by endogenous N A contributes towards an elevated breathing frequency (as they, are excitatory) when the pons is intact. At P2 and P4, on the other hand, a2 adrenoreceptors are more prevalent, 51 and their stimulation by endogenous NA contributes to a decreased respiratory frequency when the pons is present. With the application of a l and a2 antagonists to only the medulla, we attempted to determine if this was the case. If there are more medullary a 1-receptors present at PO, then Prazosin (an al-receptor antagonist) application to only the medulla, should result in a decrease in breathing frequency as it blocks the excitatory effects of noradrenaline. At P2 and P4 (where it is assumed that a2-receptors are dominant and there are fewer a 1-receptors), application of Prazosin should have less of an effect than at PO. Also, application of Rauwolscine (an a2-receptor antagonist) to the medulla at PO should have little or no effect on burst frequency if there are few medullary a2-receptors at this age. However at P2 and P4, Rauwolscine should effectively block adrenergic inputs (that bind and hyperpolarize the dominant a2 receptors) resulting in an increased burst frequency. We also hypothesized that not all adrenergic input to the respiratory rhythm generator originates in the pons. Thus, a third set of experiments was performed to determine whether there were adrenergic inputs to the rhythm generator coming from the medulla itself. There are several adrenergic sites within the caudal medulla and rostral spinal cord, namely A l / C l and A2/C2 (see Figure 1.1). It is possible that some of the effects of blocking a l and a2 receptors in the medulla of pons intact preparations could be due to blocking adrenergic inputs from sites within the medulla, rather than sites in the pons. The purpose of this experiment was to determine the relative role of these medullary sites. If they play no role, and all adrenergic input arises from the pons, then we hypothesized that we would see no change in frequency when a l and a2 antagonists were applied to the medullary preparation (ie. a preparation lacking the pons, and therefore lacking pontine inputs). 52 3.2 M A T E R I A L S AND METHODS 3.2.1 Animals and tissue preparation Sprague-Dawley rat pups were obtained from the UBC Animal Care Centre the morning of an experiment and kept warm under a heating lamp until used. The experiments were run on three different age groups of neonatal rats (PO, 2, 4) each with an n=7-10. Each animal was dissected at room temperature (~23°C) and its brain stem (pons and medulla) and spinal cord were isolated and placed in a recording dish and perfused with 27°C aCSF (preparation previously described in Chapter 2). The recording dish in this experiment (3.2.3 A and B) varied slightly as it was a split bath, in which each side could be superfused with either the same bathing solution, or differing solutions. The pons was placed on one side and the medulla and spinal cord on the other, and completely separated with a piece of perforated plastic and sealed with vasoline. This allowed us to apply drugs selectively to either the pons, or the medulla. For the first part of the experiment, both sides were perfused with regular aCSF. 3.2.2 Pharmacological Agents Several researchers have used Prazosin to block a l adrenoreceptors by bath applying it to en bloc preparations. Errchidi et al. (1991) applied 50-100 uM Prazosin for 6-9 minutes in 0 to 3 day old rats while Viemari et al. (2002, 2004) applied 50 uM to both mouse and rat preparations for 10-15 minutes. To determine the appropriate concentration to be used in this study, a quick dose response experiment was performed with concentrations of 25, 50 100 and 200 uM Prazosin. No further change in response was observed to concentrations of 50 uM and higher. Thus, to be consistent with concentrations used by other researchers, this dose of 50 uM was chosen. Yohimbine is commonly used as an a2 adrenoreceptor antagonist (Errchidi et al., 1990; 1991). The concentration typically used for bath application in neonatal rodent en bloc 53 preparations is 200 uM. We attempted to use this drug to block al adrenoreceptors in the pons at several different concentrations (50, 100 and 200 uM). Unfortunately, tonic background noise increased immensely and nerve discharge from all preparations ceased almost immediately upon drug administration. Therefore, the diastereoisomer of Yohimbine, Rauwolscine hydrochloride was used at a concentration of 200 uM. Rauwolscine binds to, and inhibits, all three types of a2 adrenoreceptors with similar affinities, and is considered a reversible antagonist (Perry et al., 1981). In addition, Rauwolscine is fifty times less potent at central a l adrenoreceptors than its diastereoisomer Yohimbine thus providing a more specific a2 adrenoreceptor antagonist (Hedler et al., 1981). Although it has a high affinity component that binds to a2 adrenoreceptors, a drawback to use of this drug is that it also expresses a low affinity for serotonin receptors (Broadhurst et al., 1988). Arthur et al. (1993) have described Rauwolscine as a partial agonist at the human serotonin 1A (5-HTiA) receptor. Although expected not to have a large effect in this study, implications of this will be discussed later. 3.2.3 Experimental Protocols A) a2-adrenoreceptor antagonist applied to the pons only, in ponto-medullary preparations Baseline activity was recorded via a suction electrode on spinal nerve C1 for 20-30 minutes. Following this, Rauwolscine was added to the aCSF bathing the pons-only to achieve a final circulating level of 200uM. The medulla continued to be superfused with regular aCSF. Flow rates on either side of the barrier were adjusted to ensure that there was no passage of drugs from one side of the dish to the other. The side being superfused with the drug was superfused at a rate of 4.5ml/min while the side superfused with aCSF was superfused at a rate of 5.5ml/min. Thus if there was any transfer of fluid between the two compartments, it would be favoured to flow in the direction toward the drug superfusate side. Activity associated with continuous drug application was recorded for 12 minutes. Early trials indicated that Rauwolscine exerted its 54 effects within the first few minutes of application, and if left on too long the signal began to deteriorate. Thus 12 minutes was deemed an appropriate amount of time to confidently record full action of the drug before damaging any cells. Drugs were washed out with regular aCSF for 12 minutes or until rhythm had almost returned to baseline frequency. At this point, the pons was transected from the medulla using fine micro-scissors. Both sides continued to be perfused with aCSF and activity was recorded for 12 minutes. B) al and a2-adrenoreceptor antagonists applied to the medulla only, in ponto-medullary preparations Baseline activity was recorded for 20-30 minutes. Following this, the aCSF bathing the medulla only was replaced with one containing either 200uM Rauwolscine or 50 uM Prazosin. Only one drug was applied in a given preparation. The pons continued to be perfused with regular aCSF. Again, flow rates were adjusted to levels indicated in experimental protocol 3.2.3A. Activity associated with continuous drug application was recorded for 12 minutes. Drugs were washed out with regular aCSF for 12 minutes, or until the rhythm had almost returned to baseline frequency. At this point, the pons was transected from the medulla using fine micro-scissors. Both sides continued to be perfused with aCSF and activity was recorded for 20 minutes. C ) al and a2-adrenoreceptor antagonists applied to the medulla only, in isolated medulla preparations The preparation was dissected out of the animal and once in the recording dish, the pons was transected at a level just rostral to the anterior inferior cerebellar artery. Baseline activity was recorded for 20-30 minutes. Following this, the aCSF was replaced with one containing either 200uM Rauwolscine or 50 \iM Prazosin. Only one drug was applied to a given preparation. Activity associated with continuous drug application was recorded for 12 minutes. 55 Recording was terminated at this step (ie. drugs were not washed out afterwards as in the two previous protocols). 3.2.4 Histology A histological verification was required to nullify the possibility of vascular or ventricular transfer of pharmacological agents from one side of the split bath to the other. This was necessary to ensure that pharmacological agents applied to one side of the split bath affected tissue only on that side of the bath. Using a similar protocol to experimental protocols A and B, horseradish peroxidase VI-A (HRP) dissolved in aCSF (1.75%) was substituted for the aCSF/pharmacological drug combination. In one experiment, the HRP flowed over the pons of a P4 preparation, while aCSF passed over the medulla for 12 minutes (to mimic protocol A). In a second experiment, the HRP flowed over the medulla of a P4 preparation, while aCSF passed over the pons for 12 minutes (to mimic protocol B). After the HRP application, both sides of the dish were drained and the brain was placed in 4% formaldehyde/PBS for 1 day then transferred to 25% sucrose (in 0.1M PBS) for 2 days. It was then placed in tissue tek and immediately transferred to a -80°C freezer for 10 minutes. Once frozen, the tissue was sliced transversely in 20 um sections. Two sequential serial slices were stored on two separate slides, while the following 13 slices were discarded. This process was repeated until the block was completely sectioned. Thus, we ended up with comparable slides with sections taken every 300 um. Using one series of slides, ~750ul of DAB/ufea/H 20 2 was puddled onto each slide and the slides were placed in a dark humid chamber for approximately 20 minutes. The slides were then rinsed in two washes of 5 minutes each in PBS. Coverslips were then mounted onto the slides with Entellan. A Nissl staining protocol was performed on the second parallel series of slides. The slides were placed in 0.1% Cresyl Violet for 5 minutes. They were then dipped in 70% ethanol (with 2-56 3 drops of acetic acid). After the sections were dry, coverslips were mounted onto the slides with Entellan. 3.2.5 Data Analysis and Statistics Two-minute samples were taken from the integrated traces and fictive breathing frequency was calculated. For control values (either pons ON or pons OFF), the samples were taken 20-30 minutes after the start of recording. Samples during drug administration (either Rauwolscine or Prazosin) were taken during the last two minutes of the twelve minute drug application. For post-transection pons OFF values, samples were taken 20-30 minutes after the transection occurred. To facilitate comparison between age groups, all values were normalized relative to control pons ON values withineach age group. To test for significance between control (either pons ON or pons OFF) and drug application or post-transection pons OFF values, paired t-tests were used on the raw data (ie. not normalized). A one-way analysis of variance (ANOVA) was performed to detect differences between all three age groups in the transection experiment, both for pons ON and pons OFF values, followed by a Tukey test. P < 0.05 was considered significant. Amplitude was not analyzed because we were unable to compare changes in amplitude in pons ON and pons OFF preparations. When the pons was transected, this often disturbed the recording electrode and altered the signal amplitude. 3.3 RESULTS 3.3.1 Histological verification Figure 3.1 illustrates two cross-sections, one from the pons (at the level of the facial nerve) and one from the medulla (at the level of the hypoglossal nerve), in an in vitro preparation that underwent HRP application to the pons only. As can be seen in the figure, HRP stained much of the tissue in a section of the pons (Fig. 3.1 A). However, no staining can be seen in the 57 cross-section of the medulla (Fig. 3. I B ) . This suggests that no drug penetrated the medulla through vascular transfer from the side of the bath containing the pons. Figure 3.1 Cross-sections (dorsal side on top) following a DAB reaction of a P4 rat in vitro brainstem spinal-cord preparation that received HRP application on the pons only. HRP stains brown. A: section taken at the level of the pons. B: section taken at the level of the medulla. Figure 3.2 also illustrates cross-sections at the level of the pons and the medulla. However these sections are from a preparation that received HRP applied to the medulla side of the split bath. Staining can be seen in cells located on the ventral and lateral surfaces of the medulla (Fig. 3.2B). No staining is seen in a section taken from the pons (Fig 3.2A) suggesting that no HRP (and therefore no drugs) were transferred through the ventricle from the medulla to the pons. 3.3.2 Effects of age and pontine inputs on fictive breathing frequency Pontine influence drastically changed between PO to P4 in rats (see Figure 3.3). At PO, the pons appeared to be excitatory as its removal resulted in a decrease in respiratory frequency (fictive breathing frequency dropped significantly from 7.5±0.4 bursts/min with the pons ON to 58 B ' # * 6 Figure 3.2 Cross-sections (dorsal side on top) following a D A B reaction of a P4 rat in vitro brainstem spinal-cord preparation that received H R P application on the medulla only. H R P stains brown. A : section taken at the level of the pons. B: section taken at the level of the medulla. 5.9±0.7 bursts/min with the pons OFF). At P2, frequency was scarcely altered after pons transection, changing from 8.0±0.5 bursts/min with the pons ON to 8.4±0.4 bursts/min with the pons OFF. Once the pons was removed in P4 rats, breathing frequency increased significantly from 6.9±0.4 bursts/min to 10.9±0.5 bursts/min. Fictive breathing frequency of ponto-medullary preparations of all three ages were comparable and were not statistically different, while breathing frequency in medullary preparations increased with age and were significantly different at all three ages. Note that breathing frequencies for the same age groups of pons ON vs. pons OFF preparations in Figures 3.4 and 3.5 do not wholly correspond to the values in Figure 3.3. This is because of a smaller sample size in Figures 3.4 and 3.5. Once the data is pooled (as in Figure 3.3), the trends become obvious and significant. 59 14 -i 0 2 4 Age group (days post-natal) Figure 3.3. Relationship between age and the effect of pontine transection on the fictive breathing frequency of the rat brainstem-spinal cord preparation. Data are presented as mean values ± S E M . A n asterisk (*) indicates a significant difference from pons O N control values within the same age group. Bars with the same letter within a treatment group (pons O N or pons OFF) were not significantly different, those with different letters were. P < 0.05 is considered significant. 3.3.3 Effects of age and pharmacological blockade ofpontine a2 receptors on fictive breathing frequency Blocking a2 adrenoreceptors in the pons had no significant effect on breathing frequency regardless of the age of the preparation (see Figure 3.4). At PO, application of Rauwolscine always resulted in a small trend toward a decrease in fictive breathing frequency (on average to 0.69±0.14 of control values) that was not significant. With P2 and P4 preparations, Rauwolscine application produced a small trend toward increases in frequency (to 1.08±0.11 in P2 and 1.12±0.11 in P4, relative to control values) that also were not significant. Removing the pons was also without effect except in PO pups where it produced a decrease in frequency. As with the Rauwolscine application, there were trends in the data following removal of the pons, and 60 although these were usually more pronounced than the changes resulting from pharmacological blockade, they were also not significant at any age other than PO. 2 5 i i ^ H pons ON (control) I I Rw applied to pons M pons OFF 0 2 4 Figure 3.4. Effect of pharmacological blockade of pontine a-adrenoreceptors with Rauwolscine (Rw) and removal of the pons at different ages in rat en bloc preparations. Data are presented as mean values ± SEM. Values are normalized as fraction of control values (ie. pons ON). An asterisk (*) indicates a significant difference from control (ie. pons ON) for each particular age. 3.3.4 Effects of age and pharmacological blockade of medullary al and a2 receptors on fictive breathing frequency, in the presence of pontine input A) al adrenoreceptor blockade Blocking a l adrenoreceptors in the medulla, in the presence of the pons, did not have a significant effect at any age (see Figure 3.5A). Prazosin decreased frequency slightly at PO, to 0.89±0.06 of control values. At P2, the drug effect was even less, as frequency decreased on average to 0.99±0.05 of control values. In contrast to the trends at PO and P2, adding Prazosin slightly increased frequency at P4, to 1.18±0.12 of control values. The changes in frequency observed once the pons was removed were similar to the overall changes seen in section 3.3.2, although these were only significant in the P4 preparations. 61 2.5 -, o 1 ? s I u_ o OD .2 J5 o i ^ B pons ON (control) I I Pr applied to medulla pons OFF pons ON (control) Rw applied to medulla pons OFF Age (days post-natal) F i g u r e 3.5. Effect of pharmacological blockade of medullary a-adrenoreceptors and removal of the pons at different ages in rat en bloc preparations. A : medullary a l adrenoreceptors were blocked with Prazosin (Pr). B : medullary a2 adrenoreceptors were blocked with Rauwolscine (Rw). Data are presented as mean values ± SEM. Values are normalized as fraction of control values (ie. pons ON). An asterisk (*) indicates a significant difference from control (ie. pons ON) for each particular age. 62 B) al adrenoreceptor blockade Blocking a2 adrenoreceptors in the medulla, in the presence of the pons, had a similar effect in all three age groups (see Figure 3.5B). Frequency was significantly decreased relative to initial baseline values when Rauwolscine was applied. The extent of the reduction in frequency, however, decreased with increasing age. In PO preparations, frequency declined to a small fraction of the initial value (0.24±0.12) when Rauwolscine was applied. In P2 and P4 preparations, this value was only 0.76±0.06 and 0.83±0.07 of the initial baseline frequency. Again, the changes in frequency observed after pontine transection were similar to the overall changes seen in section 3.3.2, although they were only significant in the P4 preparations. 3.3.5 Effects of age and pharmacological blockade of medullary al and al receptors on fictive breathing frequency, in the absence ofpontine input A) al adrenoreceptor blockade Application of Prazosin to isolated-medulla preparations always resulted in a decrease in frequency, although this was only significant in P2 and P4 preparations. Fictive breathing frequency decreased to similar levels in all three age groups: to 0.86±0.06 in PO, 0.87±0.04 in P2 and 0.89±0.04 in P4, of control values. Note that this differs from the results obtained when Prazosin was applied to the medulla and spinal cord, in ponto-medullary preparations where drug application had no significant effect. B) a2 adrenoreceptor blockade When Rauwolscine was applied to in vitro brainstem-spinal cord preparations lacking the pons, a similar trend was observed to that seen in preparations where the pons was still present (results discussed in section 3.3.4). In all three age groups, application of the a2 adrenoreceptor antagonist resulted in a significant decrease in frequency when compared to initial baseline values (note that this time, control values are recorded in the absence of pontine input). There 63 A 1 - 4 i " c 3 ^ in 8 0 8 ] W o => c m o 0.6 -I g> o > CO <D 0.4 H or Age (days post-natal) Age (days post-natal) Age (days post-natal) Age (days post-natal) Figure 3.6. Effect of pharmacological blockade of a-adrenoreceptors on rat ponto-medullary and medullary preparations at different ages. Medullary a l adrenoreceptors were blocked with Prazosin (Pr) either in the presence (A) or absence of the pons (B) . Medullary a2 adrenoreceptors were blocked with Rauwolscine (Rw) either in the presence (C) or absence of the pons (D) . Data are presented as mean values ± S E M . Values are normalized as fraction of control values (ie. pons O N in A and C, pons OFF in B and D). A n asterisk (*) indicates a difference from control (ie. pons O N in A and C, pons OFF in B and D) for each particular age. 64 was a further decrease when compared to values obtained in pons ON preparations. For example, in PO preparations where the pons was removed and Rauwolscine was applied, bursting almost always stopped, resulting in an average fictive breathing frequency of 0.026±0.026 relative to pons OFF values. However, in PO preparations with the pons intact, application of Rauwolscine did not abolish frequency, but slowed it to 0.24±0.12 relative to pons ON values. This trend was found in data from preparations of all ages. The effect of Rauwolscine on fictive breathing frequency decreased with increasing age, as was also seen in ponto-medullary preparations. 3.4 D I S C U S S I O N 3.4.1 Role of 5-HT1A in modulation the respiratory rhythm: possible confounding factor? As mentioned previously, the pharmacological agent used to block a2 adrenoreceptors is also a partial agonist at 5-HTIA (5-hydroxytryptamine receptor type 1A) receptors. Since serotonergic mechanisms are known to play a role in the control of respiratory rhythm generation, interpretation of the results of blockade with Rauwolscine must be made with due caution. Serotonergic cells are located in raphe nuclei which send out projections and release 5-HT in several respiratory-related centres including the reticular formation and phrenic nuclei (reviewed in Hilaire and Duron 1999). Generally, serotonergic activation is thought to depress respiration (Lundberg, 1980), however, depending on the method of administration and the age of the animal, serotonergic modulation can produce varying results (reviewed in Bianchi et al., 1995). While intraveneous administration of 5-HT agonists generally produced a decrease in respiration, either intracerebroventricular or in vitro administration of agonists resulted in an increase in respiratory frequency (reviewed in Bianchi et al., 1995). At the medullary level, in both neonatal rats and mice, 5-HT excited respiratory rhythm generation via 5-HTiAreceptors (Hilaire et al., 1997). At E18 in rats, 5-HTj A excitation was so potent that it was speculated to be 65 required for respiratory rhythmicity (Hilaire and Duron, 1999). The strength of 5-HT facilitation decreased with age throughout the embryonic and post-natal periods (Di Pasquale et al., 1994). Also, although 5-HT appeared to be inhibitory at the whole animal level (via 5-HTi B receptors), Edwards et al. (1990) identified an excitatory pathway through intraperitoneal injection of 5-HTIA agonists in adult rats that resulted in an increase in respiratory rate. Taken together, and with respect to our study, it appears that if Rauwolscine was having any effect as a partial 5-HT ] A agonist, it should have increased the respiratory rate in our in vitro neonatal rat preparations. Since application of Rauwolscine on the medulla always resulted in a significant decrease in respiratory frequency, any effect it was having via serotonin receptors would have diminished the overall effects of Rauwolscine on a2 adrenoreceptors. 3.4.2 Sites of catecholamine release An important finding of the present study with respect to adrenergic influences on the respiratory rhythm generator was that they were primarily medullary in origin. Alpha-1 and a2-adrenoreceptor antagonists were applied to the medulla only, in the presence and absence of the pons. If most adrenergic inputs were originating in the pons, it was expected that antagonists applied to the medulla in the absence of the pons should result in little change in breathing frequency. However, in our experiments, there was always a significant change in breathing frequency when antagonists were applied to a preparation lacking pontine input (see Figure 3.5). A) a l adrenoreceptor effects There was some small excitatory a l drive originating within the medulla at all ages as a decrease in frequency was always observed when medullary a 1-adrenoreceptors were blocked in pons OFF preparations (significant at P2 and P4) (see Figure 3.6B). When we look at a l -adrenoreceptor effects with the pons still attached, the situation changed slightly. There was no a l drive originating from the pons at PO as the blockade of a 1-adrenoreceptors with the pons off 66 or on produced the same effect (see Figure 3.6A). At P2 there appeared to be some net a l inhibition (which was offsetting the medullary excitatory a l drive), while at P4 there was even more net a l inhibition (as seen by the large difference between breathing frequency values for Prazosin application in pons ON and OFF preparations). The most likely medullary origin for the N A is the A l / C l noradrenergic neurons located in the ventrolateral medulla, along the rostral ventral respiratory group. Molecular studies presented by Hilaire et al. (presented af IUPS meeting, San Diego 2005) suggest a role for A l / C l in the modulation of the respiratory rhythm generator in mouse neonates. In c-Ret (which encodes a tyrosine kinase receptor) mutant mice, a decrease in respiratory frequency was found in in vitro preparations. In these mice, they also found that levels of NA were decreased in the pons, but not in the medulla. A decrease in cell number was found in A5 and A6, but not in A l / C l nor A2/C2 (permitting these cells to continue their release of NA). Coupled with a2-adrenoreceptor studies (see next section), it is thought that the endogenous N A originated mainly from A l / C l , and not A2/C2. B) a2 adrenoreceptor effects There was a powerful excitatory a2 drive originating within the medulla at PO (see Figure 3.6D) and this drive decreased with age. When the antagonist was targeted to a2-adrenoreceptors in a pons ON preparation, there as also a significant decrease in frequency although the extent of the frequency depression was always less (see Figure 3.6C). This suggests that not only is the excitatory a2 drive less with the pons on, but there is an inhibitory a2 drive from the pons occurring at all ages. This inhibitory drive appears to be greatest at P2 where there is the largest difference in the effect of a2-adrenoreceptor blockade when comparing pons ON versus pons OFF preparations. 67 Also, an excitatory input via a2-adrenoreceptors was found in neonatal mice (presented by Hilaire at IUPS meeting, San Diego 2005). When Yohimbine (an a2-adrenoreceptor antagonist) was applied to a pons OFF mouse preparation, frequency often decreased. It was suggested that the respiratory rhythm generator was facilitated by endogenous N A originating from A l / C l . 3.4.3 A novel scenario for the net developmental changes in pontine influence Overall, pontine actions on the medullary respiratory rhythm generator switched from providing a net excitation at PO, to a net inhibition by P4. This is clearly evident in Figure 3.3 where transection of the pons resulted in a decrease in breathing frequency at PO, no change, in frequency at P2 and an increase in frequency at P4. This finding does not agree completely with any of the proposed scenarios described in Chapter 1. We did observe an increase in pontine inhibition from P2 to P4 (similar situations were discussed in scenarios B and C). However, the observation that the pons may provide a substantial excitatory drive to the respiratory rhythm generator in PO preparations is novel. Our breathing frequency values for pons ON preparations were much higher than those reported by other researchers. Errchidi et al. (1991) recorded values of 3.8 and 5.5 bursts/min (in P0-1 and P2-4 preparations, respectively and most other researchers report comparable values to Errchidi et al. (1991) for pons ON preparations (Ballanyi et al., 1999; Ito et al., 2000; Tanabe et al., 2005; Hilaire et al., 2005). Our values were also roughly similar in all age groups: 7.5±0.4, 8.0±0.5 and 6.9±0.4 bursts/min in P0, P2 and P4 preparations respectively. If our pons ON values were replace with values similar to those reported by other groups (Errchidi et al., 1991; Ballanyi et al., 1999; Ito et al., 2000; Tanabe et al., 2005; Hilaire et al., 2005), and our pons OFF values were held the same as those we recorded, it would appear that removal of the pons always resulted in an increase in frequency in our preparations also (see Figure 3.7). An explanation for 68 our high starting values with the pons O N may lie in the improved oxygenation preparations received in our double-sided dish. Thus our pons O N preparations may have been receiving more oxygen than preparations of other researchers, which may have resulted in an increased breathing frequency. However, when the pons was removed and the mass of the preparation was reduced, our dish may then have provided no more oxygen to the medulla than other dishes, resulting in comparable breathing frequencies. This implies that other preparations are hypoxic and this alters breathing frequency and the response to pontine transection. Note that our dish was the same dish used by Zimmer, who also recorded similar high starting values. Whether the early developmental changes in the influence of the pons are partly related to oxygenation levels of ponto-medullary preparations remains to be explored. 0 2 4 0 2 4 Age group (days post-natal) Age group (days post-natal) Figure 3.7. Comparison between our recorded results and those from Errchidi et al. (1991). Results found in our study are most closely comparable to Scenario C (work of Zimmer, unpublished), where it was suggested that pontine inhibition increases with age. As noted above, Zimmer reported breathing frequencies that were similar to our current findings (8.6 and 6.74 bursts/min) in PO-2 and P4-6 pons O N preparations. Values for our P2 and P4 pons OFF 69 preparations support this scenario directly. However rather than less inhibition at PO, we found excitation. It is possible that Zimmer (unpublished) found no inhibition, rather than excitation in • her P0-P2 group because she used mostly PI and P2 pups. 3.4.4 Role of other pontine inputs to the medullary respiratory rhythm generator Since our pharmacological results cannot explain the overall effects seen when the pons was removed and how this changed developmentally, other pontine inputs must be involved besides those suggested in our original hypothesis. Several other neuromodulators may be involved, including serotonin (modulatory role discussed in section 3.4.1), neuropeptides (eg. u-opioids), and adenosine. A recent study by Tanabe et al. (2005) investigated the role of p-opioids (a type of neuropeptide) in respiratory rhythm generation in ponto-medullary preparations of PO-2 Wistar rats. While activation of opioid receptors in medullary preparations decreased respiration, application of opioid agonists to ponto-medullary preparations increased the respiratory rhythm. Tanabe et al. (2005) interpreted their results to suggest that opioids both inactivate pontine A5 noradrenergic inhibition and inhibit medullary neurons. While this scenario, as such, cannot explain what happens to the changes in the overall influence of the pons with age, it does introduce a new player in the system. Another contributor to the modulation of the respiratory rhythm is adenosine. Saadani-Makki et al. (2004) discovered a rostral pontine regulation of respiratory drive by an adenosinergic A l system. When an adenosine A l agonist was applied to ponto-medullary preparations, breathing frequency increased while blockade of these receptors resulted in a decrease in breathing frequency. Their explanation of these results involved several synaptic connections originating in the pons. They proposed that an adenosine A l agonist indirectly excites the medial parabrachial nucleus (MPB) via inhibition of presynaptic inhibitory 70 neurotransmission. As such, activation of A l receptors on a relay cell would inhibit this cell's connection (via G A B A A receptors) with the MPB. With the release of this inhibition, the MPB would then be able to excite the respiratory rhythm generator indirectly through the nucleus raphe magnus. They also observed along with other researchers (see Ballanyi et al., 1999 for review) an inhibitory effect when adenosine A l receptors were activated in medullary preparations. While roles for several of these other neuromodulators may be required to explain our current findings concerning the changing overall influence of the pons with age, at present this would require several large assumptions concerning the source of the neuromodulator in question (eg. pontine or medullary), whether or not this changes with age, and whether or not receptor distribution or neuromodulator release changes with development. For example, it is possible that at P4, there is more adenosine released or there are more A l receptors on respiratory rhythm generating cells in the medulla. Thus, if the adenosine was originating from the pons, removal of the pons would decrease this inhibitory connection, resulting in an increase in breathing frequency. Conversely, at PO, there may be more A l receptors in the pons (or more adenosine releasee there) which would further disinhibit the MPB exciting the respiratory rhythm generator when the pons was present. However, removal of the pons would eliminate this excitatory connection, resulting in a decrease in frequency as observed in PO preparations following pontine transection. As is evident, many scenarios can be hypothesized to explain a switch from an excitatory pons to one that is inhibitory. However there is little empirical evidence in the literature at present to support such changes. This is due to a lack of age-specific experiments; most researchers have not examined developmental changes in neuromodulators with respect to changes in respiratory frequency. Full explanation of this phenomenon will likely require a combination of many changes in the neuromodulatory systems (differences in the amount of 71 neuromodulator released, the number of receptors present and the location of receptors, all possibly changing after birth), none of which alone can explain all of the changes seen in pontine influence. Also, there are many other changes occurring in the brain throughout early development. The first two postnatal weeks in rats are the most dynamic in the development of brainstem respiratory nuclei in rats (Wong-Riley and Liu, 2005). There are many significant changes in expression of neurotransmitters, receptors and receptor subtypes in the first two weeks (Liu and Wong-Riley, 2002). Glutamate, glycine receptors, thyrotropin-releasing hormone, choline acetyltransferase, serotonin and norepinephrine all increase their expression with age, while GAB A, serotonin receptor IA, substance P, neurokinin 1 receptors and somatostatin decrease their expression with age (Wong-Riley and Liu, 2005). In addition, there is a developmental change in the expression of G A B A A receptor subunits in the rat Pre-Botzinger Complex (Liu and Wong-Riley, 2004). There appears to be a switch in dominance from a3 to a l subunits around PI2. Also, there is evidence for two critical developmental periods in rats. At P3-4, there is an ephemeral reduction in cytochrome oxidase (CO), glutamate and the NMDA receptor subunit 1 in the Pre-Botzinger Complex, while expression of GABA, GABAB receptors, glycine receptors and the A M P A receptor subunit 2 have a transient increase (Liu and Wong-Riley, 2002). Also over the first two weeks, the role of synaptic inhibition in respiratory rhythm generation increases and becomes important after P15 in rats and mice (Paton et al., 1994; Paton and Richter, 1995). Other developmental changes occurring include a progressive hyperpolarisation of the membrane potential (Richter and Spyer, 2001), a change in the proportion of persistent sodium current -dependent and -independent pacemaker cells (Pena et al., 2004), a cross between the membrane potential and reversal potential for chloride (resulting in an excitatory role for GABA in neonates rather than inhibitory as in adults) (Ballanyi et al., 1999; Richter and Spyer, 2001; Ritter and Zhang, 2000), and a reduction in phrenic motoneuron excitability (Greer 72 and Funk, 2005). Thus, there are many developmental changes occurring in the brainstem throughout early development and it is likely a combination of all these events that contribute to changes seen in respiratory control. 3.4.5 Role of a.2 autoreceptors in the pons The first part of the study looked at the role of pontine a2-adrenoreceptors in the modulation of breathing frequency during the first few days of life in rats. The intention of applying an a2-adrenoreceptor antagonist only to the pons was to target the a2 autoreceptors located on A5 and A6 cells. Errchidi et al. (1991) performed the same experiments in a single age group and found that blocking pontine a2-adrenoreceptors resulted in a decrease in breathing frequency. They suspected that this was due to blockade of autoreceptors located on A5 cells, which would disinhibit them, allowing for further N A to be released onto the medulla. They failed to mention how this may affect A6 autoreceptors, and what results this would have. However, Hakuno et al. (2004) found that selectively activating a2-adrenoreceptors on A6 cells resulted in a decrease in frequency in P3-4 rats as A6 cells were inhibited and no longer released NA onto excitatory medullary a 1-adrenoreceptors. No change in frequency was seen in PI-2 preparations. What we found in our experiments is not consistent with either of these previous findings (Errchidi et a l , 1991; Hakuno et al., 2004). There was no significant change in frequency in P0, P2 or P4 preparations. There was a trend, however, that was similar to the effect of pontine transection at each of these ages: a decrease in breathing frequency at P0 and an increase at P4 (see Fig. 3.4). 3.4.6 New models We always observed a decrease in breathing frequency when medullary a2-adrenoreceptors were blocked, in all ages and with or without the pons. Thus we can conclude 73 that there are a2-adrenoreceptors on medullary cells, and that they either directly or indirectly provide an excitatory stimulus to the respiratory rhythm generator when active. We therefore propose two alternative models by which this specific action is occurring (see Figure 3.8). The first model proposes that there are excitatory a2-adrenoreceptors on the respiratory rhythm generating cells. When these receptors are blocked, cells in the rhythm generator are turned " o f f and thus breathing frequency is depressed. The second model includes an intermediate cell with inhibitory a2-adrenoreceptors on its surface. This cell would normally be inhibited by endogenous N A in the medulla. However, when blocked, the cell is turned "on" and would project to an inhibitory receptor on the rhythm generator, resulting in a decreased frequency. Note that in both these models, the source for N A is within the medulla (most likely A l / C l as suggested in the figures). We favour the second model as it is more consistent with previous findings that a2-adrenoreceptors are inhibitory. This second version wil l be used in following models. Model 1 PONS MEDULLA O N O F F Model 2 PONS "OFF" MEDULLA O N ' O N " O F F ' F i g u r e 3.8. Proposed models 1 and 2 for the medullary adrenergic influence on the respiratory rhythm generator based on results from medullary a2-adrenoreceptor blockade. 74 The following model (Figure 3.9) is an attempt to interpret all of our data (both from pons transections and pharmacological experiments) concering the net influence of the pons over the first few post-natal days in neonatal rats. PONS PO P2 W f£>a1 1 smal l ,ar9e€Ta2 mediu MEDULLA P4 A6) (A5 medium ^ a l medium RRG sma © a l smal l F i g u r e 3.9. Proposed model for the medullary and pontine adrenergic influence on the respiratory rhythm generator at post-natal days 0, 2 and 4 in neonatal rats. This model is based on results from medullary and pontine a l - and a2-adrenoreceptor blockade as well as pons transections. Note that in this figure it is assumed that connections to a l -adrenoreceptors are originating from A 6 while those neurons connecting with a2-adrenoreceptors are originating from A 5 . Transection results showed a net excitation at PO which changed to a net inhibition at P4. At PO, there was no al drive while there was a small pontine a2 inhibition. In order to account for the small inhibition provided by a pons transection, the pons must have been providing an excitation via some unknown mechanism (as illustrated by a question mark in Figure 3.9). At P2, there was some net al inhibition and even more a2 inhibition from the pons. However pontine 75 transection did not alter breathing frequency thus it must be providing a strong excitatory drive to offset this adrenergic inhibition. At P4 there was even more net a l inhibition than at P2 as well as some a2 inhibition provided for by the pons. Since pontine transection results in a large excitation, this data would suggest that the unknown pontine excitatory drive provided at PO and P2 is gone. 3.5 C O N C L U S I O N S Pontine inputs changed developmentally over the first four days post-natal in rats. The pons switched from providing an excitatory input on the respiratory rhythm generation at PO, to providing an inhibitory input by P4. The pons also passed through a transitional phase at P2 where it had little influence breathing frequency. Although most research has focussed on the role of pontine noradrenergic groups A5 and A6 in modulating the respiratory rhythm, we found little noradrenergic evidence to support the changes seen in the overall influence of the pons on breathing frequency. Blocking a2-adrenoreceptors in the medulla in the presence of the pons always resulted in a decrease in breathing frequency, undermining the theory that it is through these receptors that the pons provides its inhibitory input. Also, in agreement with other studies, blocking al-adrenoreceptors in the medulla in the presence of the pons had no effect on respiratory frequency at any age. Finally, noradrenergic input appeared to be originating in the medulla and not the pons.. 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