The Breathing Brainstem Development of Inspiration

From DEPT. OF WOMEN’S AND CHILDREN’S HEALTH Karolinska Institutet, Stockholm, Sweden

DEVELOPMENT OF INSPIRATION

David Forsberg

Stockholm 2017

All previously published papers were reproduced with permission from the publisher. Published by Karolinska Institutet. Printed by US-AB © David Forsberg, 2017 ISBN 978-91-7676-880-8 The Breathing Brainstem – Development of Inspiration THESIS FOR DOCTORAL DEGREE (Ph.D.)

David Forsberg

Principal Supervisor: Opponent: Professor Eric Herlenius Professor Jan-Marino Ramirez Karolinska Institutet University of Washington School of Medicine Department of Women’s and Children’s Health Department of Neurological Surgery Division of Clinical Pediatrics Center for Integrative Brain Research Seattle Children’s Research Institute Co-supervisor: Professor Per Uhlén Examination Board: Karolinska Institutet Associate Professor Maria Lindskog Department of Molecular Biochemistry and Karolinska Institutet Biophysics Department of Neurobiology, Care Sciences and Division of Molecular Neurobiology Society Division of Translational Alzheimer Neurobiology

Associate Professor Konstantinos Meletis Karolinska Institutet Department of Neuroscience

Associate Professor David Engblom University of Linköping Department of Clinical and Experimental Medicine Center for Social and Affective Neuroscience

To Love, with hope that it will inspire you. To Josephine, for endless love and support.

ABSTRACT Breathing is essential for life, and yet we do not fully understand the mechanisms that control it. The main central pattern generators for respiration include the inspiratory generating region called the preBötzinger Complex (preBötC), and the chemosensitive region called the parafacial respiratory group/retrotrapezoid nucleus (pFRG/RTN). These are located in the ventrolateral medulla oblongata of the brainstem. To study these centers in detail, organotypic cultures of the respiratory brainstem were developed to keep the structural integrity of the neural tissue essentially intact while still allowing careful control of the microenvironment. The cultures generated synchronized respiratory network activity and motor output for up to three weeks. Characterization revealed a network organization of the respiratory regions with a so called small-world structure. These respiratory networks consists of both neurons and astrocytes. Detailed examination identified two subgroups of astrocytes. Most appeared dormant, but a subset of the astrocytes displayed persistent, rhythmic oscillating calcium activity. These active astrocytes formed an individual network, interacting with a distinct neuronal network. Furthermore, stimulation of the astrocytes increased their calcium oscillation frequency in both the preBötC and pFRG/RTN. However, while neuronal calcium oscillations in the preBötC were unaffected, they were significantly increased in frequency in the pFRG/RTN. The organotypic culture also preserved the respiratory center reactivity towards exogenous stimulus, such as opioids and hypercapnia. Hypercapnic challenge elicited a gap- junction dependent release of the inflammatory molecule prostaglandin E2 (PGE2) in the pFRG/RTN, increasing the overall network activity of this region. A release of PGE2 was also triggered by stimulation of astrocytes, which blunted a subsequent hypercapnic challenge. Thus, a new respiratory signaling pathway where PGE2 release from astrocytes following hypercapnic challenge modifies respiratory behavior to meet physiological demands was identified. In a clinical setting this might be beneficial at birth, where high

PGE2 levels set the respiratory system to perform deep breaths. However, increased levels of

PGE2 would also blunt the hypercapnic response, inducing a vulnerable period for infants. The first week after birth, cardiorespiratory dysfunction may lead to sudden unexpected postnatal collapse (SUPC), where seemingly healthy infants collapse and require resuscitation. In a retrospective study, we identified 115 SUPC cases among 313 351 live births during a 15.5-year period. Thus, the incidence of 36.7 SUPC events per 100 000 live births makes SUPC events more common than sepsis caused by group B streptococci. Seven percent of the affected children died, and about one fourth developed hypoxic ischemic encephalopathy. The majority of SUPCs occurred during the first hours after birth and where related to co-bedding, emphasizing the importance of these risk factors. Urinary PGE2 metabolite levels were high during the first days after birth when most (97 %) of SUPC events occur. These findings are important for understanding how respiratory behavior is affected during inflammatory states, such as immediately after birth and during infections, and have an impact on our ability to detect and protect against respiratory dysfunction. LIST OF SCIENTIFIC PAPERS I. Forsberg D, Thonabulsombat C, Jäderstad J, Jäderstad LM, Olivius P, Herlenius E. 2017. Functional Stem Cell Integration into Neural Networks Assessed by Organotypic Slice Cultures. 2017. Current Protocols in Stem Cell Biology, 42, 2D.13.1-2D.13.30 doi: 10.1002/cpsc.34

II. Forsberg D, Horn Z*, Tserga E*, Smedler E, Silberberg G, Shvarev Y, Kaila K, Uhlén P, Herlenius E. 2016. CO2-evoked Release of PGE2 Modulates Sighs and Inspiration as Demonstrated in Brainstem Organotypic Culture. 2016. eLife 2016;5:e14170 doi: 10.7554/elife.14170

III. Forsberg D, Ringstedt T, Herlenius E. Astrocytes Release Prostaglandin E2 to Modify Respiratory Network Activity. 2017. eLife 2017;6:e29566 doi: 10.7554/eLife.29566

IV. Herlenius E, Steinhof L*, Drevin G*, Pejovic N, Forsberg D. Sudden Unexpected Postnatal Collapse of newborn infants: Risk Factors and Role of Perinatal Transition. Manuscript

*These authors contributed equally to the work

SCIENTIFIC PAPERS NOT INCLUDED IN THIS THESIS

I. Herlenius E, Thonabulsombat C, Forsberg D, Jaderstad J, Jaderstad LM, Bjork L and Olivius P. Functional stem cell integration assessed by organotypic slice cultures. 2012. Current Protocols in Stem Cell Biology, 23, 2D.13:2D.13.1–2D.13.26 doi: 10.1002/9780470151808.sc02d13s23

II. Dyberg C, Fransson S, Andonova T, Sveinbjornsson B, Lannerholm-Palm J, Olsen TK, Forsberg D, Herlenius E, Martinsson T, Brodin B, Kogner P, Johnsen JI and Wickstrom M. 2017. Rho-associated kinase is a therapeutic target in neuroblastoma. Prococeedings of the National Academy of Sciences, USA, 114:E6603-E6612. doi: 10.1073/pnas.1706011114.

III. Meisgen S, Hedlund M, Ambrosi A, Folkersen L, Ottosson V, Ding B, Strandberg L, Ramsköld D, Forsberg D, Bergin L, Ruhrmann S, The Swedish Congenital Heart Block Study Group, Franco-Cereceda A, Hamsten A, Olsson T, Greene L, Eriksson P, Gemzell-Danielsson K, Salomonsson S, Herlenius E, Kockum I, Sonesson SE, Wahren-Herlenius M. Auxilin is a novel susceptibility gene for congenital heart block that directly impacts fetal heart function. Manuscript

POPULÄRVETENSKAPLIG SAMMANFATTNING Att andas är absolut livsviktigt. Dessutom använder vi andningen i en rad andra beteenden, till exempel när vi pratar, luktar, hostar, nyser och äter. För att både kunna genomföra den automatiska in- och utandningen och samtidigt utöva medveten kontroll över rörelserna krävs ett avancerat styrsystem. Detta system finns i en del av hjärnan som kallas hjärnstammen, där många grundläggande kroppsfunktioner kontrolleras. Andningen koordineras av ett flertal nätverk av nervceller som samarbetar för att skapa rytmiska andningsrörelser och anpassa dessa efter situationerna som kroppen befinner sig i. Huvuduppgiften är att kontinuerligt syresätta blodet och andas ut den koldioxid som produceras av kroppens celler.

Den här avhandlingen har foksuerat på andningens grundläggande kontrollmekanismer och hur andningen anpassas när koldioxinivåerna i blodet stiger. För att undersöka aktiviteten i andningsnätverkens etablerade vi en metod som medför att en del av hjärnstammen från en mus kan hållas vid liv i en skål i flera veckor. Under denna tid fortsätter nätverken att skicka nervsignaler som om nerverna fortfarande är kopplade till muskler i musen. Med metoden är det möjligt att studera andningskontroll under längre tid och undersöka hur andningen påverkas av till exempel läkemedel eller långvarig inflammation.

Vi använde metoden för att studera nervcellsnätverkens struktur och hur de påverkas av inflammation och förhöjda koldioxidnivåer. Båda nätverken som studerades visade sig vara organiserade som så kallade ”small-world networks”. Detta innebär att nervcellerna är organiserade i grupper som ligger nära varandra och att en del av dessa celler kopplar samman de olika grupperna. Denna struktur gör nätverket stabilt och motståndskraftigt mot skador och linkande strukturer kan ses hos t.ex. nätverk av flygplatser i världen och bland grupper av vänner på Facebook.

I nästa steg undersökte ví hur de här nätverken påverkas av prostaglandin E2. Prostaglandin

E2 är en molekyl som frisätts i inflammatoriska processer och bland annat ger upphov till feber. Sedan tidigare vet vi att molekylen kan påverka andningen och orsaka andningsuppehåll hos nyfödda barn med infektioner. Vi visade att prostaglandin E2 kunde påverka hjärnstammens andningsnätverk. I ett av nätverken hämmade prostaglandin E2 aktiviteten, vilket förklarar effekterna vi sett tidigare hos både möss och människor. Däremot i det andra nätverket vi studerade var effekten den motsatta. Aktiviteten ökade i nätverket som också är det nätverk som ökar andningen när koldioxidnivåerna stiger i blodet. För att undersöka det nätverket närmare utsatte vi det för förhöjda koldioxidnivåer. Detta ledde till att aktiviteten ökade som förväntat, men också till att prostaglandin E2-nivåerna steg.

Vi misstänkte att prostaglandinet frisattes från en särskild typ av nervceller kallade astrocyter. När vi testade att aktivera astrocyterna ökade nätverkets aktivitet samtidigt som prostaglandin

E2 nivåerna steg. Alltså verkar den inflammatoriska molekylen prostaglandin E2 frisättas från astrocyter som en del i svaret på höga koldioxidnivåer, vilket också skulle kunna förklara varför andningen hos småbarn är påverkad i samband med infektioner. Dessutom spelar prostaglandin E2 en viktig roll i samband med förlossningen, dels för att skydda hjärnan vid den tillfälliga syrebrist som uppstår och dels för att förbereda barnet på att ta djupa andetag.

Nivåerna av prostaglandin E2 stiger under förlossningen tillsammans med en rad andra stresshormoner. När prostaglandin E2-nivåerna är höga de första dagarna i livet är koldioxidsvaret försämrat, vilket kan leda till att barnet kollapsar om andningen påverkas, istället för att andningsaktiviteten ökar.

Vi har visat att denna typ av plötslig spädbarnskollaps hos tillsynes friska barn är vanligare än tidigare befarat. Incidensen är också nästan dubbelt så hög som den för blodförgiftning orsakad av en viss bakterietyp kallad grupp B-streptokocker, vilket är en fruktad och välkänd sjukdom. I vår studie kunde vi också visa att majoriteten av kollapserna inträffar under det första levnadsdygnet, och i synnerhet de första 4 timmarna efter förlossningen. Detta är under den period då prostaglandin E2-nivåerna är som högst. Att sova i samma säng som det nyfödda barnet visade sig vara en riskfaktor för att barnet ska kollapsa, precis som man tidigare befarat. För att skydda barnen direkt efter födseln, bör riktlinjer för omhändertagande av nyfödda under det första dygnet och främja säker hud-mot-hud-kontakt i vården etableras.

CONTENTS 1 Background ...... 6 1.1 The Respiratory System and complexity of the brainstem ...... 6 1.1.1 The preBötzinger Complex ...... 6 1.1.2 The Parafacial Respiratory Group/Retrotrapezoid Nucleus ...... 8 1.1.3 Postinspiratory Complex and the Triple Oscillator Model ...... 10 1.1.4 Modulatory Regions ...... 10 1.1.5 Hypoglossal Nucleus ...... 11 1.2 Glia ...... 11 1.2.1 Microglia ...... 11 1.2.2 Astrocytes ...... 11 1.3 Methods for studying the respiratory system ...... 12 1.3.1 In Vivo ...... 12 1.3.2 Brainstem-Spinal Cord Preparation ...... 13 1.3.3 The Transverse Medullary Slice ...... 13 1.3.4 Organotypic Cultures...... 14 1.3.5 Selecting the Technique ...... 14 1.4 Chemosensitivity ...... 14 1.4.1 Carbon Dioxide ...... 15 1.5 Inflammation ...... 16 1.5.1 Prostaglandins ...... 17 1.6 Clinical Aspects ...... 17 1.6.1 Apneas ...... 18 1.6.2 Congenital Central Hypoventilation Syndrome ...... 18 1.6.3 Sudden Infant Death Syndrome ...... 18 1.6.4 Sudden Unexpected Postnatal Collapse ...... 19 2 Aims ...... 21 3 Methods ...... 22 3.1 Subjects ...... 22 3.1.1 Animals ...... 22 3.1.2 Humans ...... 23 3.2 Respiration in vivo ...... 23 3.3 Organotypic slice cultures ...... 23 3.4 Calcium Time Lapse imaging ...... 24 3.5 Electrophysiology ...... 25 3.6 Optogenetics ...... 25 3.7 Molecular biology techniques ...... 26 3.7.1 Immunohistochemistry ...... 26 3.7.2 Propidum Iodide ...... 27 3.7.3 Enzyme-linked Immunosorbent Assay ...... 27 3.7.4 Quantitative Reverse Transcriptase Polymerase Chain Reaction ...... 27 3.7.5 Liquid Chromatography Tandem Mass Spectrometry ...... 27

3.8 Data analysis and statistics ...... 27 3.8.1 Plethysmography ...... 27 3.8.2 Calcium Time Lapse Imaging ...... 28 3.8.3 Statistics ...... 29 3.9 Investigation of SUdden Unexpected Postnatal Collapse ...... 29 4 Results and Discussion ...... 33 4.1 Study I and II – The Breathing Brainstem ...... 33 4.2 Study III – Breathing by Glue ...... 38 4.3 Study IV – Sudden Unexpected Postnatal Collapse ...... 39 5 Conclusions ...... 43 6 Future Perspectives ...... 45 7 Acknowledgements ...... 47 8 References ...... 49

LIST OF ABBREVIATIONS aCSF Artifical Cerebrospinal Fluid ALTE Apperent Life-Threateing Event ATP Adenosine Triphosphate BötC Bötzinger Complex cAMP Cyclic Adenosine Monophosphate CCHS Congenital Central Hypoventilation Syndrome COX-2 Cyclooxygenase 2 Cx Connexin DAMGO [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin Dbx1 Developing Brain Homeobox 1 DIV Days In Vitro EEG Electroencephalography Egr2 Early Growth Response 2 ELISA Enzyme-Linked Immunosorbent Assay EPR Eicosanoid Prostanoid Receptor e-pF Embryonic Parafacial Oscillator FLRF Phe-Leu-Arg-Phe-amide

FR Respiratory Frequency GBS Group B Streptococcus Gfap Glial Acidic Fibrillary Protein GFP Green Fluorescent Protein GPCR G-protein Coupled Receptor Hcn Hyperpolarization-activated Cyclic Nucleotide-gated Channel HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic Acid HIE Hypoxic Ischemic Encephalopathy Iba1 Ionized Calcium Binding Adapter Molecule 1 2+ ICAN Ca -activated Nonspecific Cation Currents ICD-10 International Classification of Diseases, 10th revision IHC Immunohistochemistry IL-1β Interleukin 1β + INaP Voltage-dependent Persistent Na Current

InsP3 Inositol 1,4,5-triphosphate Kcc2 K+/Cl- cotransporter 2

Kcnk potassium channel subfamily K Kcnq potassium channel subfamily Q Map2 Microtubule Associated Protein 2 MeCP2 Methyl-CpG Binding Protein 2 mPGES-1 Microsomal Prostaglandin E Synthase 1 MrgA1R Mas-related Gene A1 Receptor + - NBCe1 Electrogenic Na /CO3 Cotransporter 1 Ncx Na+/Ca2+ Exchanger Nk1R Neurokinin 1 Receptor NTS Nucleus Tractus Solitarius pCO2 Partial Pressure of CO2 pO2 Partial Pressure of O2 PCR Polymerase Chain Reaction pFL Lateral Parafacial Region pFRG/RTN Parafacial Respiratory Group/Retrotrapezoid Nucleus pFV Ventral Parafacial Region PG Prostaglandin Phox2b Paired-like Homeobox 2b PiCo Post Inspiratory Complex preBötC preBötzinger Complex qRT-PCR Quantitative Reverse Transcriptase Polymerase Chain Reaction ROI Region of Interest RTN Retrotrapezoid Nucleus S100β S100 Calcium-binding Protein β SIDS Sudden Infant Death Syndrome SUDI Sudden Unexpected Death in Infancy SUEND Sudden Unexpected Early Neonatal Death SUPC Sudden Unexpected Postnatal Collapse Tuj1 Neuron-specific Class III β-tubulin

U-PGE2m Urinary PGE2 Metabolite VE Minute Ventilation VGlut2 Vesicular Glutamate Transporter 2

VT Tidal Volume

“Complexity is only apparent owing to incomplete knowledge”

Robert Huckstepp and Nicholas Dale, 2011

1 BACKGROUND

1.1 THE RESPIRATORY SYSTEM AND COMPLEXITY OF THE BRAINSTEM Respiratory control is often recognized as complex, mainly because we have yet to discover the involved parts, both molecular and anatomical (1). Almost two millennia ago, Galen, a physician tending to the gladiators in ancient Rome, suggested that the nervous tissue above the lower neck needs to be intact for continuous breathing (2, 3). However, a first milestone in respiratory research came in the late 1700s, at the time of the French revolution. Then the French physician Julien-Jean-César Le Gallois understood that mammals do not need their heads to breathe. Le Gallois identified a respiratory center in the medulla oblongata (located in the caudal brainstem) and showed that the removal of this caused the cessation of breathing (4). This also provided the first evidence of a specific function related to a specific anatomic region within the brain (4, 5). During the 19th century, this particular area was more closely defined and Santiago Ramón y Cajal suggested that several aggregations of cell bodies (nuclei) within the brainstem affected the respiratory rhythm (6). The work was continued further by Curt von Euler by confirming the rhythmogenic properties of the brainstem and suggesting the presence of central chemosensitivity (7-10). Later, Hugo Lagercrantz, together with Curt von Euler, investigated respiratory control in the neonate (8, 11-14).

Apart from generating the vital rhythmic breathing movements that maintain blood oxygen

(O2), carbon dioxide (CO2) and pH levels, the system is also necessary for many vocal and emotive behaviors, e.g. talking, crying, yawning, vomiting, sneezing and sniffing (15). To understand these more complex behaviors, we first need a deeper understanding of the mechanisms behind the basic rhythmicity (3).

In essence, the machinery controlling breathing can be divided into two major parts; the rhythm generators and the modulators. For rhythmogenesis, three centers within the medulla oblongata have been defined; the preBötzinger Complex (preBötC) (16), the parafacial respiratory group/retrotrapezoid nucleus (pFRG/ RTN) (17) and, more recently, the Postinspiratory Complex (PiCo) (18). The Bötzinger complex (BötC), Nucleus Tractus Solitarius (NTS), rostral ventral respiratory group (rVRG), caudal ventral respiratory group (cVRG), raphe nucleus as well as pontine circuits, including the Kölliker-Fuse nucleus and the lateral parabrachial region, provide input to and modulate the rhythmogenic regions (19).

1.1.1 The preBötzinger Complex The preBötC was identified and first characterized in 1991 and is located in the ventrolateral medulla oblongata (16). Its importance in generation of rhythmogenesis has been shown repeatedly. First, the preBötC is necessary as well as sufficient for the generation of inspiratory output (15, 16). Second, transection of the brainstem rostral to the preBötC does not abolish the respiratory activity. However, what remains is inspiratory dominated (19). Third, preBötC specific lesions creates an ataxic breathing pattern that eventually ceases (20) and optogenetic inhibition of the preBötC interrupts inspiratory rhythm (21). Thus, there is evidence that the preBötC is the kernel for inspiration.

The mechanistic details of how this inspiratory rhythm is generated still remain a mystery. A hypothesis is the presence of pacemaker neurons, as pacemaker properties are found within many other biological rhythmogenic processes, such as circadian rhythms, in cardiac cells of the sinus node in the heart and cyclic contraction of the pylorus (22-25). The discovery of neurons with pacemaker-like properties in the brainstem led to the identification of the preBötC and several neurons in this region displays oscillatory activity (16, 26). However, only a subset of them are voltage-dependent (16). Initially, the voltage-dependent persistent + Na current (INaP), was thought to be the only driver of pacemaker activity (27). Later, it was discovered that blocking Ca2+ channels, specifically Ca2+-activated nonspecific cation current

(ICAN), also affected the pacemaker properties (28-30). If both INaP and ICAN are inhibited simultaneously, the respiratory rhythm generated in the preBötC stops. However, this effect can be reversed by a general excitation of the region, indicating that the pacemaker mechanisms are not essential for respiration (29).

Instead, a respiratory network is suggested to generate the rhythm – the group-pacemaker theory (31, 32). Here, a subset of neurons fire tonically at a low rate. This activity then increases via positive feedback mechanisms during the pre-inspiratory phase and then results in the inspiratory burst (15, 27). Some of the neurons in the group-pacemaker complex are spontaneously active, and subsequently excite silent neurons through synaptic transmission.

These can then propagate the signal further as positive feedback. INaP and ICAN are still involved in the process, as synaptic excitation cause a Ca2+ release from intracellular stores, inducing ICAN activity (27), and INaP maintains the membrane potential at near spike threshold (33).

Figure 1. The breathing brainstem. Within the medulla oblongata, several neural networks collaborate to generate rhythmic respiratory output. The main central pattern generators include the preBötzinger Complex (preBötC), the parafacial respiratory group/retrotrapezoid nucleus (pFRG/RTN) and the Postinspiratory Complex (PiCo). In addition, there are several modulatory regions; the Bötzinger complex (BötC), Nucleus Tractus Solitarius (NTS), the rostral ventral respiratory group (rVRG), the caudal ventral respiratory group (cVRG), the raphe nucleus (RN), the Kölliker-Fuse nucleus (KF) and the lateral parabrachial region (LBPr). XII; hypoglossal nucleus, VII; facial nucleus, V; trigeminal nucleus. Adapted from Smith et al. (19) and Anderson et al. (18).

Several cell types of the preBötC are involved in the rhythmogenic network. However, the core is made up of neurons whose precursors express the homeodomain transcriptions factor Dbx1 (Dbx1 neurons). Specific optogenetic inhibition of Dbx1 neurons results in apnea (21). A subgroup of the Dbx1 neurons express the neurokinin 1 receptor (Nk1R, encoded by Tacr1). Tacr1 is primarily expressed in, but not limited to, this region in the brainstem and has been used together with somatostatin as a marker to identify the preBötC (34-37). Nk1R is the receptor for substance P that increase respiratory frequency (34, 38). Furthermore, Nk1R neurons are necessary for the generation of respiration (39). Another important characteristic of the preBötC is the presence of µ-opioid receptors, that a§re co-expressed with the Nk1R. Activation of these receptors inhibit rhythmic preBötC activity (34).

During the embryonic period, the emergence of the preBötC prepares the fetus for a life dependent on breathing (40). In mice, Dbx1 is expressed between embryonic day E9.5 and E12.5 (41, 42) and expression of Tacr1, together with spontaneous rhythmically organized neural activity, emerge at E15.5 (43). The timing of the process is however species specific (36, 43). When preBötC rhythmicity emerge, fetal breathing movements are generated. Before this, however, when no organized rhythmic activity is seen, neuronal activity can still be measured in the preBötC region (44).

Although much of the mechanistic details of inspiratory rhythm generation remains to be discovered, it is evident that the preBötC plays the major role in respiratory control.

1.1.2 The Parafacial Respiratory Group/Retrotrapezoid Nucleus Spontaneous rhythmic activity is found in a region ventral to the facial nucleus about 24 hours prior to the emergence of the preBötC (in mice) (40). This was termed the embryonic parafacial oscillator (e-pF) (44). During early stages, this activity entrains the PreBötC rhythm and is synchronous to preBötC activity, but later develops into a pre-inspiratory pattern (44, 45). The neurons within the e-pF are derived from transcription factor Egr2- expressing cells that later start to express the transcription factor Paired-like Homeobox 2b (Phox2b). Thus, they are of a different origin compared to the preBötC cells. The early activity is dependent on functioning INaP, similar to the preBötC, and the intercellular synchronization is disrupted by inhibition of gap junctions (43, 46).

The overall rhythmic respiratory activity is not dependent on the e-pF during development. However, inactivation of e-pF reduces breathing frequency significantly, since it drives the preBötC rhythm (46, 47). The e-pF develops into what, in the neonatal period, is known as the parafacial respiratory group (pFRG). Part of this development is suggested to be affected by the change in intracellular chloride levels (48). Thus, the e-pF, and later pFRG, constitutes a second rhythmogenic region that, like the preBötC, expresses Tacr1 and is subsequently stimulated by substance P (46, 49). However, in contrast to the preBötC, the pFRG does not express µ-opioid receptors (44) and is therefore not inhibited by opioids. Furthermore, the expression of Phox2b is essential as a genetic deletion of Phox2b early in development results in death shortly after birth due to respiratory failure (50).

In the adult rodent, an anatomically similar region was initially discovered as a central chemosensitive area on the ventral medullary surface (51-54). After exposure to increased

CO2 levels (hypercapnia), the area showed increased levels of c-fos mRNA (an immediate early gene) (55). Further anatomical characterization defined an extension towards the trapezoid nucleus, and thus the region was named the Retrotrapezoid Nucleus (RTN) (56). These discoveries were made in parallel with the identification of pre-inspiratory neurons in the neonatal pFRG (57-60). Later, the pFRG and RTN was shown to overlap (61-63). Although the pFRG and RTN have slightly different functionality due to the developmental differences they do display similar chemosensitive properties. Therefore, they are commonly referred to as the pFRG/RTN and the differences are likely due to developmental factors (62).

The pFRG/RTN has several important properties. It contains pre-inspiratory neurons that fire before inspiratory bursts and then again during the post-inspiratory period. Thus, these neurons have been termed biphasic (49, 64-66). They express both Phox2b and Tacr1 (49) and have intrinsic burst-generating properties and will continue to burst even after synaptic blockade (67). Phox2b is essential for pFRG/RTN development and PHOX2B mutations cause Congenital Central Hypoventilation Syndrome (CCHS, see 1.6.2) in humans (68, 69). Specific lesioning of pFRG/RTN Phox2b-neurons in adolescent rats does not inhibit breathing, but severely reduces the ability to respond to CO2 (70, 71). Similarly, inhibition of the pFRG/RTN reduces hypoxic and hypercapnic responses without affecting the eupneic (normal) breathing pattern (72).

Furthermore, it has been suggested that the pFRG/RTN is mainly responsible for active expiration and modulation of the rhythmic frequency (15). Optogenetic stimulation of pFRG/RTN neurons results in an increased respiratory activity, both through increased respiratory frequency and tidal volume (73-75). This property is mainly related to the lateral part of the pFRG/RTN (pFL). Activating the pFL results in an increased active expiration at rest (73, 76), while hyperpolarization at rest does not affect respiration (76). Furthermore, pFL is silent at rest, due to tonic GABAergic and glycinergic inhibition (76, 77). Removing this inhibition also results in increased activity, which is apparent in neurons called late- expiratory neurons. These progress to abdominal, hypoglossal and laryngeal motoneurons. The late-expiratory neurons do not express Phox2b (77).

In contrast to the pFL activity, the ventral part of the pFRG/RTN (pFV) is active at rest and provides an excitatory drive to breathe (76, 78, 79). The pFV is also involved in increasing respiratory drive during hypercapnia, low levels of O2 (hypoxia) and exercise (76). This contributes to the pFRG/RTN being the most important chemosensitive region that serves to adapt the respiratory rhythm to the levels of O2, CO2 and pH in the blood. This chemosensitivity (see 1.4) is strongly related to Phox2b expressing pre-inspiratory neurons that are intrinsically sensitive to both CO2 and pH (49, 80, 81). Hypercapnia may also evoke a disinhibition of the non-chemosensitive late-expiratory neurons of the pFL, resulting in increased active expiration (77).

The pFRG/RTN and the preBötC are interconnected; glutamatergic preBötC neurons project to the pFRG/RTN and glutamatergic pFRG/RTN neurons project to the preBötC (15). A tight collaboration between these two regions is necessary for functional respiration (82). As mentioned previously, the respiration-related brainstem system emerges with the development of the e-pF (44) that later transform into the pFL (82). Both the preBötC and the pFL have central pattern generating abilities and can oscillate independently of each other (46). Postnatally, the preBötC takes over the central pattern generation as respiration becomes dominantly inspiration-related with passive expiration (83). The pFL of the pFRG/RTN then loses its ability to drive respiration alone (82)

1.1.3 Postinspiratory Complex and the Triple Oscillator Model A third rhythmically active respiration-related network within the medulla oblongata has recently been identified and baptized as the Postinspiratory Complex (PiCo). In addition to the inspiration of the preBötC and the active expiration of the pFRG/RTN, the PiCo generates a post-inspiratory rhythm, firing after the inspiratory preBötC bursts. Located between the pFRG/RTN and the preBötC (see Figure 1), PiCo is connected to the preBötC via GABAergic pathways, responsible for synchronizing the activity of the different regions. It is also likely that the PiCo receives stimulating input from the pons as PiCo needs norepinephrine to maintain its activity in vitro. PiCo likely plays a role in synchronizing the respiratory cycle and in the coordination with post-inspiratory behavior such as swallowing and vocalization (18).

1.1.4 Modulatory Regions

1.1.4.1 Bötzinger Complex and the Kölliker-Fuse Nucleus The BötC mainly provides inhibitory input to the preBötC, reducing muscle tonus during expiration (84) and possibly contributing to the tonic inhibition of the pFL (77). Ablation of the BötC, as well as inhibition of the Kölliker-Fuse nucleus, leads to a lack of post-inspiratory activity (84-86). The Kölliker-Fuse nucleus is suggested to be a relay station for sensory processing in the respiratory apparatus (87, 88). Projections from the Kölliker-Fuse reaches both into the preBötC and the Bötzinger Complex (54, 56). Connections between the Kölliker-Fuse nucleus and the PiCo are also likely to exist, but are yet to be found (18). The BötC and the Kölliker-Fuse nucleus collaborate to coordinate respiratory phases (89).

1.1.4.2 Nucleus Tractus Solitarius The NTS receives input from mechanosensors in the lung and chemoreceptors in the carotid bodies via vagal afferents (90). Its caudal part also contains chemosensitive neurons expressing pH-sensitive K+ channels and Phox2b (91), similar to the pFRG/RTN. These neurons also increase their activity during hypoxia and hyperoxia (92). The NTS mainly projects to the Kölliker-Fuse nucleus (88).

1.1.4.3 Raphe nucleus and serotonergic neurons The raphe nucleus is a region related to chemosensitivity, similar to the pFRG/RTN and the caudal NTS (93). Raphe lesions cause an attenuated chemosensitive response as well as a temporary inhibition of respiratory output due to a loss of serotonergic neurons (94, 95).

Although the serotonergic neurons of the raphe nucleus are not the cells responsible for CO2 sensing (96) they have an important role in modulating respiratory frequency (15, 96, 97). The raphe neurons project to both the pFRG/RTN and preBötC and increases overall respiratory activity through the release of substance P and serotonin (79, 96, 98, 99).

1.1.5 Hypoglossal Nucleus The hypoglossal nucleus of rodents is involved in breathing since it provides respiration- related motor output to the muscles of the tongue and pharynx (100, 101). Some of the premotoneurons of the hypoglossal nucleus originate in the preBötC (102), but the majority comes from the intermediate medullary reticular formation which is related to several orofacial behaviors such as sniffing and vocalization (102-104). The preBötC premotoneurons express Dbx1 during development (101).

1.2 GLIA Neuroglia were described more than 150 years ago (105, 106) and includes astrocytes, oligodendrocytes, microglia and NG2+ cells (107-109). Up until 2010, almost 90 % of scientific publications on neural cell types only concerned neurons (108), but now a more detailed picture of the role of glia in the brain is emerging (109, 110).

Oligodendrocytes, who myelinate the brain (108, 111) and NG2+ cells, or polydendrocytes, whose function within the brain remains to be determined (Sic!) (107, 112) are at present not thought to be involved in respiratory control.

1.2.1 Microglia Microglia are the resident immune cells defending the brain against microbes (113) and ischemic stroke (114) and are involved in the inflammatory machinery after brain damage (115). As inflammation is known to affect respiration and is connected to respiration-related diseases such as sudden infant death syndrome (SIDS) and apneas (116-121), there is increasing interest in investigating microglia in relation to breathing. It has been shown that microglia can modulate respiration and affects the ability to autoresuscitate. However, similar effects were seen both after inhibition and activation of microglia, raising some concern on their specific role (122).

1.2.2 Astrocytes The astrocytes, or star-like cells, named by Lenhossék in 1895 (123), have a vast number of functions within the brain. As the most abundant neuroglia, they provide structural and metabolic support for neurons and are responsible for the homeostasis of the brain (110, 124). Synapses in the brain are surrounded by astrocytic perisynaptic processes that are critical for

genesis, maturation and maintenance of neuronal connections (124, 125), partly through signaling via the gap junction Connexin (Cx) 43 (126, 127). Furthermore, astrocytes facilitate the glutamate-glutamine cycle is necessary for the function of glutamatergic neurons (124). Thus, astrocytes fulfill diverse functions in the brain, and recent findings suggest a functional division into distinct subtypes (128).

Furthermore, astrocytes are excitable in the sense of Ca2+ mediated excitability (129) due to the presence of Ca2+ pumps and Ca2+ release channels in the endoplasmic reticulum membrane (130). They also express various metabotropic receptors that couple to the inositol 2+ 1,4,5-triphosphate (InsP3) pathway and induce a release of Ca from the endoplasmic reticulum (131) and exocytosis of transmitters to the extracellular space (109, 129, 132-137). The astrocytes may also signal among themselves through gap junction-mediated Ca2+ waves (110, 130). This signaling system organizes astrocytes in a glial networks (6).

The astrocytes also have an important role in the respiratory system (138). Glia specific toxins depress breathing in vivo (139) as well as preBötC activity in vitro (140). Three subpopulations of astrocytes, with different membrane properties related to voltage- dependent currents and glutamate transporters, have been described in the preBötC (141). Two of them exhibited low activity during electrophysiological recordings, but the third group expressed outwardly rectifying K+ channels providing rhythmic properties (142). The presence of rhythmically oscillating astrocytes within the preBötC was later confirmed (141, 143-145). These astrocytic oscillations have been suggested to be pre-inspiratory (144, 145).

Notably, the investigations of the role of astrocytes in preBötC respiratory rhythm generation have been performed in neonatal animals. Thus, an overlap of undifferentiated cells and cells identified as suggested astrocytes is possible (146). Therefore, the development of astrocytic activity throughout maturation warrants further investigation (143, 147). However, there is no debate about astrocytes playing an integral role in central chemosensitivity (see 1.4).

1.3 METHODS FOR STUDYING THE RESPIRATORY SYSTEM Respiratory control is difficult to study since the complex multicomponent machinery that constitutes the respiratory apparatus lies deep within the brainstem. In vivo models reproduce the complexity of the system, but provide little insight to the specific neural networks. In vitro models, on the other hand, allow the study of respiratory centers and the specific cells within the neural networks. However, these in vitro preparations often exclude important modulatory inputs. Most studies of the neural networks that govern respiration have used models such as the brainstem-spinal cord (“en bloc”) preparation (66, 148-150) or transverse medullary slices (35, 149, 151).

1.3.1 In Vivo Compared to the initial experiments during the 19th century, modern techniques have made more detailed studies in vivo possible. Respiratory rhythm can be studied using a plethysmograph chamber (152) or by electrical recordings of nerve activity (153).

Furthermore, transgenic and optogenetic approaches provide in vivo modulation of specific cells (21) and mouse strains can be used as disease models in which respiration is affected (68). The main advantage of in vivo techniques is that the intact system is studied, including all brainstem nuclei, carotid bodies and diaphragm movements. However, working in vivo entails limited access to the neural networks and techniques to circumvent this problem are still being developed (21, 121). There are also semi-in vivo models, e.g. a rabbit model, where the respiratory apparatus is intact but the cerebrum is removed (154, 155). Furthermore, it is preferable to investigate basal rhythm generation under calm circumstances, which are difficult to obtain in a plethysmograph (156). Another option is the use of electromyography or nerve recordings, which are precise but require anesthesia, which modulate breathing (21, 121, 157). Therefore, due to both technical and ethical reasons, studies of respiration under physiological conditions are close to impossible.

1.3.2 Brainstem-Spinal Cord Preparation The brainstem-spinal cord preparation is a model in which the brainstem and spinal cord are isolated and kept in vitro in a hyperoxygenated, bicarbonate based buffer solution mimicking the cerebrospinal fluid (artificial cerebrospinal fluid, aCSF) (59, 60, 100, 158). From the preparation, extracellular recordings from the cranial and cervical spinal nerves can be performed as well as individual neuron patch-clamping (100, 148, 159). Although lacking input from cortical regions or areas outside of the central nervous system, the brainstem- spinal cord preparation contains a rather complete apparatus for central respiratory control and rhythm generation (159, 160). This allows studies of the pontine-medullary interactions, with connections between the pFRG/RTN and the preBötC intact (60, 161). The main issue with the technique is that the respiratory-related rhythm is artificial and heavily dependent on the experimental conditions (100). Furthermore, the brainstem-spinal cord preparation only provide limited access to the respiratory networks.

1.3.3 The Transverse Medullary Slice The rhythmic, transverse medullary slice preparation is the most used in vitro method for studying respiratory centers (162). The brainstem is isolated from neonatal mice (or rats) and submerged into aCSF and then sectioned transversally to generate slices containing respiratory regions. Immediate (“acute slice”) experiments are performed directly after sectioning under temperature control and continuous perfusion of aCSF (16). In the slice, respiratory networks and neurons are available for electrical recordings, either extracellular or patch-clamping, and Ca2+ time-lapse imaging (162).

Even more so than the brainstem-spinal cord preparation, the generated respiratory-related rhythm of the medullary slice is artificial. Furthermore, the medullary slice lack several modulatory regions and instead isolate a few respiratory-related networks (162). This promotes studies of specific cells or centers that can determine a high level of mechanistic details, but also results in a loss of inter-center connections (16). This affects the rhythm- generation properties and the modulation of them, which has to be taken into account when

interpreting data (84, 85, 89, 162). There has also been a debate whether this artificial respiratory rhythm is related to normal breathing (eupnea) or gasping (32, 163, 164). However, as results obtained in vitro converge with in vivo data, it is safe to say that the rhythm of the transverse medullary slice preparation is eupnea-related (162).

1.3.4 Organotypic Cultures An aspect of both the brainstem spinal-cord preparation (see 1.3.2) and the acute transverse medullary slice (see 1.3.3) is that they only remain active in terms of rhythm generation for some 5-6 hours (16, 100). With a K+ concentration of 9 mM, the activity can be maintained for more than 8 hours (165). Still, it is impossible to study development and long-term effects on respiratory rhythm under such settings.

Organotypic slice cultures preserve the three-dimensional structure of neural tissue, allowing functional circuits to be studied and manipulated over time (166-169). Initially, the system was developed for hippocampal tissue (170), but the organotypic cultivation method has lately been used in studies of the cerebellum (171) and the brainstem (172, 173). In the organotypic culture, the slice is provided necessary nutrients and oxygen, keeping the tissue alive for several weeks (170, 174-178). An effect of this cultivation is flattening of the tissue. The cells migrate and spread, reducing the slice thickness over time. Yet, during flattening, intercellular connections are preserved and therefore organotypic cultures improves the optic conditions (167, 176, 177).

1.3.5 Selecting the Technique In summary, there is no perfect technique for studying the respiratory apparatus. All models come with advantages and disadvantages. Therefore, careful study design and a combination of several techniques often generates the most reliable results (121, 152, 156). It is also important to evaluate the experimental setup when comparing studies, since small variations might cause huge discrepancies in the results. For example, changing the extracellular concentration of Ca2+ or K+ will alter the respiratory-related frequency and its reaction to environmental stresses (165, 179). Even slight differences in experimental temperatures and developmental stage may generate conflicting results if not taken into account during interpretation (180). In addition, different slice thickness of transverse medullary slices will give different degrees of oxygenation within the tissue (181). Thus, a multitude of variables has to be considered and carefully set to meet the specific aims of the study as well as provide proper generalizability.

1.4 CHEMOSENSITIVITY

Cells consume O2 to utilize energy in various cellular processes. This leads to the production of CO2 as a waste product (182). The main, and vital function of respiration is to keep blood pO2 and pCO2 levels relatively constant to maintain cellular function and body homeostasis.

Recognizing and adjusting respiration based on pO2 and pCO2 levels is known as chemosensitivity. This response involves both central (confined within the brain) and

peripheral regions (such as the carotid bodies) (183). Lack of oxygen or retention of CO2 has depressant effects on the central nervous system and ultimately leads to unconsciousness and death (184-186). As the hypoxic response has not been investigated in this thesis it is not discussed here. For more information, please see e.g. the recent review by Gourine and Funk (187).

1.4.1 Carbon Dioxide

1.4.1.1 The hypercapnic response Hypercapnia induces a rapid and distinct alteration of the respiratory rhythm. It leads to increased respiratory activity with elevated breathing frequency and an increased tidal volume (152). This reduces the CO2 concentration in the lungs, leading to removal of CO2 from the blood. Simultaneously, O2 levels are kept high, facilitating the formation of CO2 - + from HCO3 and H in the blood (188), making it accessible for ventilation.

1.4.1.2 Mechanisms behind the central hypercapnic response We are yet to fully understand the mechanisms behind the central hypercapnic response (6, 62). Several brainstem regions are involved, including the pFRG/RTN (189, 190), the raphe nucleus (191) and the NTS (92). The main central chemosensitive region is the pFRG/RTN, even though the importance of serotonergic input from the Raphe nucleus (see 1.1.4.3) is debated (94).

Within the pFRG/RTN there are Phox2b expressing pre-inspiratory neurons that are intrinsically sensitive to changes in CO2 levels and pH (192). This is partly due to a pH mediated inhibition of potassium channels, which depolarizes the neurons (193, 194). The potassium channel subfamily K (Kcnk) has several members linked to chemosensitivity who are expressed by the chemosensitive neurons of the pFRG/RTN (93, 195, 196). For example, Task-2 channels (encoded by Kcnk5) are inhibited during hypercapnia and genetic ablation of these channels both increases basal activity of the pFRG/RTN in vitro and reduces the hypercapnic response in vivo (195). Another potassium channel subfamily that is involved in sensing CO2 and acidification is the Kcnq (93, 197). These are regulators of neuronal membrane excitability and widely expressed in the brain (93). Specifically, Kcnq2 has been shown to be involved in respiratory control and animals lacking this channel die of respiratory failure within 24 hours after birth (198). Kcnq2 together with Task-2 and a third potassium channel, Thik-1 (encoded by Kcnk13), limit activity of pFRG/RTN neurons under resting conditions. Furthermore, hyperpolarization-activated cyclic nucleotide-gated (Hcn) channels (a cation channel) increase the Kcnq channel conductance (199). High CO2 levels, causing an increase in H+ concentration, will inhibit Task-2 resulting in a depolarization. This inhibits Hcn channels, while the Kcnq channel activity increases causing further depolarization (93). Additionally, the Kcnq channels are downstream targets of the serotonergic input to the pFRG/RTN (197).

The involvement of astrocytes in the hypercapnic response have emerged the last decade 2+ (147, 153). An increase of Ca concentration in astrocytes follows slight acidification. This leads to a vesicle-independent ATP release (153, 189, 200), which then acts on purinergic receptors on pre-inspiratory neurons (153, 201). ATP has also been suggested to be released through the opening of Cx hemichannels (1). Some Cxs (Cx26, Cx30 and Cx32) are directly modified by CO2 (202, 203). E.g., CO2 induce the formation of carbamate bridges in the Cx26 protein structure, inducing an open state (203).

The involvement of astrocytes in chemosensitivity is further supported by evidence of increased respiratory activity (similar to the hypercapnic response) after stimulation of pFRG/RTN astrocytes (138, 153). Furthermore, the clinical condition Rett syndrome features a reduced response to increased pCO2 levels, likely due to a dysfunction in astrocytic methyl- CpG binding protein 2 (MeCP2). Mice lacking MeCP2 are used as a model for Rett syndrome and have a reduced CO2 sensitivity. Notably, re-introduction of the MeCP2 gene selectively in astrocytes rescues the normal respiratory phenotype. Thus, a major part of the blunted hypercapnic response is due to astrocytes inability to react to changes in pCO2 and pH (204).

2+ The astrocytic increase in Ca concentration after acidification is likely due to activation of + - + the electrogenic Na /CO3 cotransporter NBCe1 (134). The NBCe1 cotransports Na and - + + 2+ HCO3 into astrocytes. Increased Na concentration drives a Na /Ca exchanger (Ncx), 2+ causing an increase in Ca concentration, which leads to activation of downstream signaling pathways. The NBCe1’s activity increases with intracellular acidification (134) and is - dependent on extracellular HCO3 (205). Furthermore, this mechanism is dependent on a membrane potential kept at a level that facilitates the action of the Ncx (134). The inwardly rectifying K+ channels Kir4.1 is involved in membrane maintenance in astrocytes (206) and genetic ablation of the channel reduces central chemosensitivity (207).

Furthermore, the inflammatory molecule prostaglandin E2 (PGE2) and the eicosanoid prostanoid 3 receptors (EP3Rs) are involved in the CO2 response (152).

1.5 INFLAMMATION Inflammation is the process generated by the body’s immune system in response to damage (208). During inflammation, various signaling molecules are released to mobilize the immune cells. Cytokines, for example interleukin 1β (IL-1β), induce the production of prostaglandins, such as PGE2. PGE2 is produced by the enzyme microsomal prostaglandin E synthase-1

(mPGES-1) from prostaglandin H2 (PGH2), which is generated from arachidonic acid, mainly by cyclooxygenase 2 (COX-2) (208). There is substantial evidence connecting an inflammatory state to apneas in the neonatal period (156, 209). This respiratory depression is attenuated by COX-2 inhibition (118, 150). In addition to the direct effects on basal breathing, activation of the inflammatory system affects the respiratory response to hypoxia, anoxia and hypercapnia (152).

1.5.1 Prostaglandins Prostaglandins, together with thromboxanes, constitute the prostanoids which is a group of eicosanoids that are produced from arachidonic acid by COX enzymes. There are four prostaglandins among the prostanoids; PGE2, PGD2, PGF2 and PGI2. For PGD2 there are two receptors and one receptor for each of PGF2 and PGI2, while several exists for PGE2. The

PGE2 receptors are named eicosnoid prostanoid (EP) receptors 1, 2, 3 and 4 (210, 211). The different EPRs are GPCRs, with, in total, eight corresponding GPCRs (210, 212). This allows the diverse actions of PGE2 (210). Furthermore, the EP3R, due to mRNA splicing, exists in at least four different isoforms the mouse; EP3A, EP3B, EP3C and EP3D (212).

1.5.1.1 Prostaglandin E2

PGE2 have been showed to affect neural networks in the brain. For example, PGE2 can induce fever as a response to inflammation (213). As blood-brain barrier endothelial cells express mPGES-1 the systemic inflammation can be transferred to the brain. IL1-β can activate mPGES-1 which induces the production of PGE2 (213, 214). The PGE2 is then released on the brain side of the barrier (156, 215).

Furthermore, PGE2 also affects the brainstem and modifies respiration, mainly by depressing breathing activity. This has been shown in several different species, including rodents (156), sheep (216) and humans (116). The effects of PGE2 on breathing are seen already during the fetal period when PGE2 inhibits the fetal breathing movements. Furthermore, inhibition of

PGE2 synthesis increase fetal breathing movements (216). Even during the neonatal period,

PGE2 can induce hypoventilation and apneas (156, 216).

More recent studies have increased the knowledge on the mechanisms underlying the PGE2- induced respiratory depression (121, 156). The actions of PGE2 are mediated via actions on EP3Rs (156). Furthermore, mice lacking the EP3R fail to respond adequately to hypercapnia (152).

1.6 CLINICAL ASPECTS Breathing is vital. Thus its regulation and control is of paramount interest. Furthermore, diseases related to respiration, such as respiratory infections, chronic obstructive pulmonary disease and cancers within the respiratory system, is the second most common cause of death worldwide (217). A well-known clinical example is the adverse effect of opioids which causes respiratory depression (218) directly connected to the expression of µ-opioid receptors in the respiratory networks of the brainstem (34). There are also several respiratory disorders, some of which have known mechanistic backgrounds while others are still unexplained. Below are descriptions of the clinical settings of dysfunctional breathing that are relevant to this thesis.

1.6.1 Apneas Apneas are respiratory pauses, defined as e.g. a reduced respiratory rate of at least 10 seconds and tidal volume less than 16 % of the average tidal volume (219). These are often associated with bradycardia and desaturation of infants (220). Apneas can be caused by obstruction of the upper airways, as in obstructive sleep apneas (221), or they can originate from dysfunctions in respiratory control (central apnea) (222). The central apneas are strongly linked to prematurity and the number and durations of apneas are correlated with the gestational age of premature infants (220, 222). There is also a correlation with birth weight, where lower birth weight is related to more frequent apneas (220). An increase in apnea frequency is also associated with infection (116, 222). In some recent studies, it was shown that changes in respiration and heart rate might precede the clinical symptoms of infections. Thus, events with apneas, bradycardia and desaturation might be predictive of sepsis (116,

222-224). The mechanism behind infection-induced apneas is likely the effect of PGE2 on the respiratory centers of the brainstem (see 1.5.1) (116, 156). The acute consequences of apneas are related to ensuing desaturation and bradycardia. This may lead to hypoxic-ischemic damage to the brain and even death (220).

1.6.2 Congenital Central Hypoventilation Syndrome A rare clinical condition is CCHS, or Ondine’s curse. The disorder is related to the genetics of the rhythmogenic regions (15). An alanine expansion in the PHOX2B protein leads to a dysfunctional breathing apparatus with a blunted CO2 response (50, 68, 225). Dysfunction of the protein generates the same respiratory problem in both mice and humans (226, 227). This has led to increased understanding of the functionality of Phox2b neurons (1.1.2) and the hypercapnic response (see 1.4.1.1). The clinical characteristics of the disease is a severe central apnea during sleep, which, in the absence of external stimuli or ventilatory assistance, causes death (228).

Notably, there are several cases of patients with mutations in PHOX2B, which present later in life with typical CCHS symptoms triggered by upper airway infections (229). We speculate that the PGE2-pathway induced by infection might be involved in the emergence of autonomic dysfunction. Thus, similar mechanism as in neonates are likely relevant to adults (68, 116, 119, 156).

1.6.3 Sudden Infant Death Syndrome SIDS is a syndrome where an infant dies of an, per definition, unknown cause. However, the syndrome is regarded to be multifactorial and a relation to cardiorespiratory failure is thought to be a common factor in a majority of cases (119, 230-232). The triple risk model suggests that the pathogenesis involves three parts; a vulnerable infant, a critical period in development and external stressors (233-235). The vulnerable infant suggests that abnormalities in neurologic and/or autonomic function precedes SIDS. The critical period of development relates to that SIDS occur during the period when autonomic control is still developing and has yet to reach a mature, more robust function. Thirdly, an external stressor,

such as prone position during sleeping, obstructed airways and high temperature is needed to cause the collapse that leads to SIDS (230, 233, 236-238).

Mechanistically, SIDS has been related to serotonergic dysfunction. This has been suggested to be part of the vulnerable infant (239). Disruption of serotonin signaling may cause prolonged neonatal apneas (15, 240) and some SIDS cases have adverse serotonin levels compared to post-mortem controls (239, 241, 242). It has also been suggested that there is a relation between SIDS and PHOX2B and its role in the development of autonomic control (242) as PHOX2B polymorphisms are more frequent in SIDS cases compared to controls (243). Moreover, infection and activation of the immune system may alter the infant’s arousal mechanisms, promoting cardiorespiratory dysfunction (116, 219, 235),

1.6.4 Sudden Unexpected Postnatal Collapse Sudden Unexpected Postnatal Collapse (SUPC, Figure 2) is considered a rare but potentially deadly or neurologically catastrophic cardiopulmonary failure occurring in seemingly healthy infants (244). However, there is no clear consensus on terminology, impairing comparability between studies. The definitions of several similar clinical conditions such as apparent life- threatening event (ALTE), brief resolved unexpected event, sudden unexpected early neonatal death (SUEND), sudden unexpected death in infancy (SUDI) and SIDS overlap with each other and with SUPC (245).

The aetiology of these conditions remains unknown, but several risk factors have been proposed. These include a primiparous mother, prone position, early unsafe skin-to-skin contact (SSC), co-bedding, unsupervised breastfeeding, low socioeconomic status, maternal smoking, and use of mobile phones the first hours after birth (245). The triple risk model could, just as with SIDS (see 1.6.3), explain the setting of SUPC (231, 246).

The outcomes of the SUPC-related conditions are even less well-studied. In the most catastrophic cases, they result in death of the infant (SUDI, SUEND, SIDS). In several cases, a cause of death (and collapse) were identified; including Group B Streptococcus (GBS) infection, congenital heart disease and metabolic disorders. Among survivors, 17 to 60 % suffered from neurological sequelae at discharge (245).

Figure 2. Sudden Unexpected Postnatal Collapse. During a sudden unexpected postnatal collapse, the infant becomes cyanotic and hypotonic and requires resuscitation. Despite being a potentially catastrophic condition, little is known about aetiology, risk factors and even incidence. Photograph taken by father, used with permission.

2 AIMS The overall aim of this thesis is to investigate neural networks responsible for the control of respiration in a setting related to breathing disorders of infants. Specifically, the aims were:

I) To investigate the plasticity of brainstem neural circuits and their activity and responsiveness to micro-environmental changes during early development II) To investigate the role of astrocytes in breathing rhythm generation

III) To study the effect and role of prostaglandin E2 on the central respiratory activity during the neonatal period IV) To characterize the clinical setting of Sudden Unexpected Postnatal Collapse and its relation to respiratory network activity in the brainstem

3 METHODS Several laboratory techniques have been utilized in this thesis (see Table 1). For detailed descriptions, please see the respective studies.

Table 1. Techniques utilized in this thesis.

Technique Usage (Studies I-IV) Plethysmography Measures respiratory activity in vivo (II) Organotypic Slice Cultures Keeps neural tissue alive in vitro (1-III) Ca2+ time-lapse imaging Detects variations in Ca2+ concentration over time (1-III) Electrophysiology Measures electrical activity in cells and tissue (II-III) Optogenetics Uses of light to control cell activity (II) Immunohistochemistry Detects proteins in cells (I-III) Enzyme-linked Immunosorbent Assay Measures molecule concentrations in samples (II-III) Quantitative Reverse Transcriptase Detects and quantifies the expression of genes (II) Polymerase Chain Reaction Liquid Chromatography Tandem Mass Detect and quantify levels small molecules (IV) Spectrometry

3.1 SUBJECTS

3.1.1 Animals In study 1-III mice were utilized mice to investigate the basic mechanisms behind respiratory rhythm and its modulation. All animals were reared by their mothers under standardized conditions with a 12:12-hr light-dark cycle and food and water was provided ad libitum. The experiments have been performed in accordance with European Community Guidelines and approved regional ethic committees. As the wild-type base, we utilized C57 black (C57BL/6J) inbred mice. Based on these, two different transgenic mouse strains were bred.

3.1.1.1 B6.E14Tg2a-Ptger3tm1Unc

To investigate the signaling pathway of PGE2, the EP3R gene (Ptger3) was selectively deleted to generate knockout mice (Ptger3-/-). The EP3R was chosen since it has been reported to be involved in modification of respiration previously (152, 156). Apart from weighing more, the phenotype of Ptger3-/- mice is very similar to normal C57BL/6J mice.

3.1.1.2 B6.Cg-Tg(hGFAP-tTA::tetO-MrgA1)1Kdmc/Mmmh To identify and investigate astrocytes and their role in respiratory networks, a mouse strain where green fluorescent protein (GFP) was expressed under the GFAP promoter was created. Offspring from GFAP-tTA and tetO-Mrgpra1 were crossed to generate the double transgenic B6.Cg-Tg(hGFAP-tTA::tetO-MrgA1)1Kdmc/Mmmh. These mice were designed to also express the mas-related gene A1 receptor (MrgA1R) in Gfap expressing cells. The MrgA1R is activated by RF-amide neuropeptides that are involved in nociception (247). Normally, the receptor is not present in the brain (248), which means that this transgenic mouse model

allows for Gfap-expressing cell specific activation by addition of for example Phe-Leu-Arg- Phe-amide (FLRF) (249, 250).

3.1.2 Humans In study IV, human subjects were investigated retrospectively during a 15.5-year period, from 2002 to 2017, in seven delivery wards and neonatal centers in Stockholm, Sweden; Karolinska University Hospital Solna, Karolinska University Hospital Huddinge, Danderyd Hospital, Sachs’ Children and Youth Hospital, BB Södertälje, BB Sophia and BB Stockholm. These centres account for around a quarter of all births in Sweden. The cohort was selected from all 313 351 live births during that period. Furthermore, a prospective study, including normal term or late preterm infants as well as infants exhibiting sudden unexpected postnatal collapse from 2011 was done. Medical records in the ante-, peri-, and neonatal chart registries Obstetrix (Siemens, Munich, Germany) and TakeCare (Compu Group Medical, Phoenix, AZ, USA) were searched for ICD-10 (International Classification of Diseases, 10th revision) diagnoses relating to severe cardiorespiratory and asphyxia events. Premature infants born before 35 weeks of gestations or infants with a registered umbilical artery pH <7.0 or BE <- 12 at birth were excluded.

3.2 RESPIRATION IN VIVO Breathing in live and awake animals (9 days old) was measured using a dual-chamber plethysmograph. Here the mouse is contained in a sealed chamber, while its nostrils and mouth have their openings in a second chamber. In this way, the gas mixture the mouse breathes can be closely controlled, and every breath causes a pressure change in the sealed chamber containing the body. These pressure changes are measured and translated to gas flow during ventilation. From the gas flow data, respiratory frequency and tidal volume were calculated. During experiments the mice were exposed to normal air and hypercapnic challenge (5% CO2 and 20% O2 in N2).

In a subset of animals, PGE2 was, prior to the mouse being placed in the plethysmograph, injected into the lateral ventricles of the brain (intracerebroventricular injection) under anesthesia. In control animals, only vehicle (aCSF containing in mM; 150.1 Na+, 3 K+, 2 2+ 2+ − − - Ca , 2 Mg , 135 Cl , 1.1 H2PO4 , 25 HCO3 and 10 glucose) was injected. After experiments, the brain was examined at the injection site. When visible intracranial bleeding was present, the animal was excluded from analysis.

3.3 ORGANOTYPIC SLICE CULTURES The majority of the data presented in study I, II and III, were obtained using organotypic slice cultures of respiratory brainstem regions from 3-day-old mice pups. The mice pups were decapitated and the brain dissected out in dissection medium (55% Dulbecco’s modified Eagle’s medium, 0.3% glucose, 1% HEPES buffer and 1% Penicillin-Streptomycin). The brain was then sectioned into 300-µm-thick transverse slices using a McIllwain Tissue Chopper.

Subsequently, slices containing the respiratory regions were identified by anatomical landmarks. The preBötC was located by the presence of the hypoglossal, ambiguous, trigeminal and solitary tract nuclei, as well as the inferior olives and medullary pyramids (Figure 1). This is well described in (165, 179, 251). The pFRG/RTN was easily localized by the presence of the facial nucleus.

Selected slices were transferred to semi porous insert membranes. Here, nutrients are provided through hydrophilic driven diffusion of medium (containing 55% Dulbecco’s modified Eagle’s medium, 32.5% Hank’s balanced salt solution, 0.3% glucose, 10% fetal bovine serum, 1% HEPES buffer and 1% Penicillin-Streptomycin) placed underneath the membrane and oxygenation of the slices occurs by contact with the gas above the membrane (the Stoppini Interface method) (175).

During cultivation, medium was replaced every second day and the brainstem slice cultures were kept for 7 to 21 days in vitro (DIV) before various experiments were performed.

3.4 CALCIUM TIME LAPSE IMAGING In this thesis, the relative changes in intracellular Ca2+ concentration were measured using fluorescent Ca2+ binding dyes; Fluo-4 AM and Fura-2 AM. Fura-2 AM is a ratiometric Ca2+ dye, meaning it can be used to measure the exact intracellular Ca2+ concentration. However, as the dynamics of spontaneous activity in neural cells is not heavily dependent on the exact concentration of Ca2+, this property of Fura-2 AM was not employed in the studies included here. Instead, Fura-2 AM was used as it is excited by light in the ultraviolet spectrum and thus does not overlap with green fluorescent protein (GFP) present in for example Gfap expressing cells of the B6.Cg-Tg(hGFAP-tTA::tetO-MrgA1)1Kdmc/Mmmh mice. Fluo-4 AM is a non-ratiometric dye, but compared to Fura-2 AM it displays a higher difference in fluorescent intensity between Ca2+ bound dye and non-bound dye.

Both dyes were dissolved in dimethyl sulfoxide and pluronic acid, and mixed with aCSF + + 2+ 2+ - - - (containing in mM: 150.1 Na , 3 K , 2 Ca , 2 Mg , 135 Cl , 1.1 H2PO4 , 25 HCO3 and 10 glucose). As the dyes were in an AM form, meaning dyes are linked to an acetoxymethyl ester, bath loading was used to get the dye into the cells. The acetoxymethyl ester makes the dye uncharged and thus lipid soluble so that it can penetrate the cell membrane. Once inside the cell, esterases will cleave the acetoxymethyl ester, leaving a charged molecule trapped inside the cell.

When Ca2+ binds to the dyes, the fluorescent intensity increases. This was measured through epifluorescence wide field imaging using a Zeiss Axioexaminer D1 microscope. Images were captured using an EMCCD camera over time, creating a series of frames where the relative changes in Ca2+ concentration could be visualized and used as a measure of neural cell activity.

3.5 ELECTROPHYSIOLOGY Electrophysiology includes techniques where the electrical activity of living tissue is measured. It ranges from recordings of single ion channel activity using patch clamp, to clinical diagnostics of whole brain activity using electroencephalography (EEG) in human patients. In study II, two types of electrophysiological methods were assessed. To investigate the electrical characteristics of neurons in the organotypic slice culture, whole-cell patch clamp recordings were made. With whole-cell patch clamp, a micropipette, containing an electrode in form of a metal wire connected to an amplifier, is used to puncture the cell membrane, giving the electrode access to the cell’s internal environment. Thus, the sum of all electrical activity across the membrane can be investigated. To visualize cells prior to patching, infrared-differentiated contrast microscopy was used. The pipettes had an initial resistance of 5–10 MΩ in a solution containing in mM: 110 K-gluconate, 10 KCl, 10 HEPES, 4 Mg-ATP, 0.3 guanosine triphosphate and 10 phosphocreatine. All recordings were performed in current-clamp mode, meaning the current was kept constant while the voltage was monitored. Electrical properties, including input resistance, membrane time constant and action potential threshold, were evaluated after the recordings.

To investigate neuronal respiratory-related motor output from the preBötC slice, hypoglossal nerve activity and hypoglossal nucleus neuronal population discharges were recorded. Such extracellular field potential recordings measure the local field potential that is generated by the synchronized neuronal activity in the region of interest. As the hypoglossal nucleus have respiratory-related motor neuron activity in rodents, this region can be used to measure the respiratory motor output (252).

3.6 OPTOGENETICS Optogenetics is a recently developed technique that utilizes light to open light sensitive ion channels (253, 254). These ion channels, such as channelrhodopsins and halorhodopsins, open when they are exposed to light of a specific wavelength. Channelrhodopsins are opened by blue light and then become permeable to cations, inducing a depolarization of the cells that express them. Halorhodopsins, on the other hand, are opened by red light and become permeable to chloride ions, thus hyperpolarizing the cells that express them. Several different and specific variants of channelrhodopsins and halorhodopsins exist, making fine tuning of individual electrical cell activity possible (254, 255).

In study II, we utilized the halorhodopsin Halo57, which is opened by light with a wavelength around 625 nm. Expression of Halo57 was controlled by the Ptger3 promoter and thus only expressed in cells also expressing EP3R. Its gene was delivered using a lentiviral vector. Organotypic brainstem slice cultures were moved to a biosafety level 2 laboratory and exposed to the lentivirus at 1 day in vitro. After 5 days, viral transfection was complete and Halo57 was expressed in EP3R positive cells, allowing specific inhibition of these cells. During Ca2+ time lapse imaging, a separate, custom-built light-emitting diode system was

used to deliver light through an optic fiber cable to a cannula that was pointed towards the specific region of the brainstem slice.

3.7 MOLECULAR BIOLOGY TECHNIQUES

3.7.1 Immunohistochemistry Immunohistochemistry (IHC) is a technique in which a target antigen, such as a protein or a small molecule, is detected in a tissue by using an primary antibody raised against this antigen (256). This primary antibody can then be visualized directly and indirectly in different ways. In the included papers of this thesis we used indirect visualization that employed a secondary antibody coupled to a fluorophore targeting the primary antibody. This allowed for imaging of labelled cells directly under during epifluorescence and laser scanning confocal microscopy.

The use of IHC in organotypic slice cultures is somewhat different compared to the more extensively used thin cryosections. Based on a previously published protocol (256), IHC labelling was employed to investigate the expression of several neuronal and glial markers within the slice. Antibodies against several proteins were used to target specific cells. Neuron-specific class III β-tubulin (Tuj1), K+/Cl- cotransporter 2 (Kcc2) and microtubule associated protein 2 (Map2) were used to identify developing and differentiated neurons. To find glutamatergic neurons specifically, an antibody against vesicular glutamate transporter 2 (VGlut2) was used. The presence neurokinin 1 receptors identified respiratory neurons within the preBötC and the pFRG/RTN and the Phox2b expression identified the pFRG/RTN region. The distribution of labelled cells following IHC with antibody targeting EP3R within the respiratory regions was also investigated. Antibodies targeting glial acidic fibrillary protein (Gfap) and S100 calcium-binding protein β (S100β) were used to locate astrocytes while an antibody targeting ionized calcium binding adapter molecule 1 (Iba1) located microglia. For detection of gap junctions, antibodies targeting Cx26, Cx32 and Cx43 were used. Finally, to identify apoptotic cells an antibody against cleaved caspase3 was used.

The organotypic slice cultures were fixed in paraformaldehyde, permeabilized with a mixture of Tween20 and Triton X100 and subjected to blocking with bovine serum albumin. Then primary antibodies against above mentioned proteins were applied for 36-48 hours at 4 °C. A secondary antibody against the immunoglobulin G structure of the primary antibody was then applied for 1,5 hours at RT. imaging of labelled cells were performed using epifluorescence and laser scanning confocal microscopy.

In this thesis, IHC was used to locate protein expression, and no value has been given to differences in fluorescence intensity. Quantification was limited to stained or non-stained cells and therefore counted as positive or negative.

3.7.2 Propidum Iodide In the initial characterization of the organotypic slice culture, propidium iodide was used to investigate the extent of cell death. Propidium iodide binds and stains DNA and RNA but does not pass over intact cell membranes and thus only stains necrotic and apoptotic cells (257).

3.7.3 Enzyme-linked Immunosorbent Assay Enzyme-linked Immunosorbent Assay (ELISA) is a test that through color shifts identifies the presence of a substance (258). During subsets of Ca2+ time-lapse imaging experiments, aCSF was collected continuously and immediately frozen on dry ice and stored at -80°C. The samples were later used for ELISA analysis to measure to the concentration of PGE2, which allowed the PGE2 levels around the brainstem slice culture to be monitored during the experiments.

3.7.4 Quantitative Reverse Transcriptase Polymerase Chain Reaction As the EP3R exists in different transcript isoforms due to post-transcriptional splicing (212) quantitative Reverse Transcriptase polymerase chain reaction (qRT-PCR) was employed to investigate the proportion of the different transcripts in the preBötC and the pFRG/RTN. The respiratory regions were dissected out from the brainstem slice, and pooled litter-wise to reduce the effect of tissue piece size. After RNA isolation, cDNA was synthesized and the reverse transcription performed. Relative quantification values were calculated using the 2- ΔΔC T method (259).

3.7.5 Liquid Chromatography Tandem Mass Spectrometry Urine samples were collected from healthy term or near term babies as well as from babies after a SUPC. The samples were stored at -80 °C for later analysis with liquid chromatography tandem mass spectrometry to examine the content of the major urinary metabolite of PGE2, tetranor-PGE2 metabolite (117). All samples were analyzed in duplicates. Initially, the samples were spiked with deuterated internal standards and acidified. The results were compared with external standard curves.

3.8 DATA ANALYSIS AND STATISTICS

3.8.1 Plethysmography In vivo plethysmography generates high-resolution data of airflow over time. As this is accomplished by measuring small changes in pressure, large artefacts are created when the mouse moves. Therefore, all recordings were visually analyzed and only periods of stable respiration, without movement artefacts were selected for further analysis. Mean respiratory frequency (FR; breaths/min), tidal volume (VT) and minute ventilation (VE) were calculated from the airflow data. Volume measurements (VT and VE) were analyzed as volume related to body weight. Sighs, defined as breath with larger amplitude and a biphasic inspiratory phase, were excluded from the analysis of FR, VT, VE, but counted separately.

3.8.2 Calcium Time Lapse Imaging To analyze cell-specific data from Ca2+ time lapse imaging experiments, regions of interest (ROI) for all active cells were selected with a semi-automated method. From the raw data, the standard deviation of the experimental period for each pixel was calculated. This created an image of cells with oscillating Ca2+ concentration. This could then be used to detect the ROIs. For each ROI, mean fluorescence intensity and x- and y-coordinates were extracted from the raw data.

To investigate cell-cell interactions and synchronized network activity, linear similarity was quantified between two waves as one is shifted in time (Pearson correlation) (260). The individual Ca2+ activity traces were compared pairwise for each cell pair, and for each pair a correlation coefficient was calculated. This data was summarized in a correlation matrix, which was converted to an adjacency matrix through the use of a cut-off level. Thus, cell-cell correlation was considered either correlated or not correlated. The specific value of the correlation coefficient was not analyzed further in this thesis. The cut-off was selected by a procedure where the true Ca2+ signal was scrambled, producing correlation coefficients that are random based on a scrambled time frame, without affecting the overall activity. The 99th percentile of a set of scrambled experiments was used as cut-off, leaving only 1% of the correlation coefficients above cut-off dependent on chance. In study III, this method was refined and the 99th percentile for the scrambled version of each experiment was used for its respective true data set. Thus, the cut-off was individualized for each network providing more accurate information.

Data on pairwise cell-cell correlation was used to visualize the network structure by plotting a line between the cell coordinates. Network connectivity was calculated by dividing the number of connected cells with the total number of cells. Network structure was analyzed by calculating the mean shortest path length (λ) and mean clustering coefficient (σ), as well as the proportion between these (small-world parameter). The clustering coefficient is the number of neighbors of a node that are also neighbors of each other, divided by the total number of possible links between the neighbors. The shortest path length is the minimum number of nodes needed to be passed when travelling from one node to another.

In nature, a network can be either regular or random. In a regular network, all nodes (e.g. neural cells) is connected to the same number of other nodes (261). In a random network, nodes are connected in a random fashion. Small-world networks combine features of both regular and random networks, with a short intermodal distance (low mean shortest path length) as in random networks and high degree of clustering (high clustering coefficient) as in regular networks (262). The small-world structure of networks is found in many biological systems, as it provides a structure that is suited for regional specialization and efficient signal transfer. These properties are necessary for brain functions and hence this is a common organization of neural networks (263).

In study II and III, many analyses and comparisons are based on Ca2+ oscillation frequencies. With spontaneous neural activity, the concentration of Ca2+ will vary with cell depolarization and hyperpolarization, creating an oscillatory pattern. As these oscillations represent cell signaling, the oscillation frequency represents cell activity. The Ca2+ time-lapse imaging traces were normalized individually by calculate the ΔF/F0, where ΔF=F1−F0. F1 is the specific fluorescence intensity of a cell at a specific time point, and F0 is the average intensity of 30 s before and after F1. The frequency was calculated using a Fourier transform (264).

3.8.3 Statistics Statistical analysis of paired comparisons was performed by Student’s t-test when data was normally distributed, and by Mann-Whitney’s U-test for non-parametric data. Full factorial two-way analysis of variance was performed when there was more than one independent variable or multiple observations, followed by Student’s t-test post-hoc. All tests were analyzed as two-sided and in all cases p<0.05 was considered statistically significant. No data sets were compared more than 20 times; hence no statistical corrections were made.

3.9 INVESTIGATION OF SUDDEN UNEXPECTED POSTNATAL COLLAPSE From the identified cohort (see 3.1.2), infants > 35 weeks of gestation, with good postnatal adaption (10 min Apgar score ≥8, pH >7 and BE >-12) and no indications of perinatal asphyxia, with a SUPC that required resuscitation during the first 7 days of life were included. These infants were identified using personal identification numbers. Their medical journals were examined to determine inclusion or exclusion. Patients not fulfilling the inclusion criteria or having any congenital malformation or a probable cause of the collapse were excluded. Thus, only seemingly healthy infants were included. Furthermore, cases where details around the event was missing or no medical personnel’s testimony could be obtained were excluded. Cases found in multiple searches were only included once. See figure 3 for details on inclusion and exclusion. For each case, a number of details on pregnancy and birth, the event and discharge were identified from the medical records (see Table 2). The different parameters were used to characterize the SUPC and identify risk factors through a comparison with the Swedish birth registry and a cohort of patients diagnosed with early neonatal sepsis caused by group B streptococci (GBS). To further study eventual underlying mechanisms, a prospective study was performed where inflammatory variables including urinary tetranor-PGE2-metabolite (U-PGE2m) were analyzed from healthy infants and infants exhibiting SUPC events (117).

Figure 3. Inclusion flow chart for SUPC cases. 313 351 infants were born alive during the study period. Of these, 474 possible SUPC cases were identified using ICD-10 diagnosis screening. 68 cases of group B streptococci sepsis were identified from the same cohort. Initially, 267 cases were excluded to limited data on the event or no collapse during the first 7 days of life, i.e. the screened diagnoses were of another origin. 184 cases of collapses remained and 10 were excluded because of perinatally identified causal factors, e.g. congenital malformations. One case was excluded due to maternal drug use during pregnancy. An additional 59 cases were identified in more than one search and only included once. This resulted in 115 cases of SUPC.

Table 2. Parameters identified in medical records of SUPC cases.

Pregnancy and Birth Event Discharge/Follow up Apgar score Age Neurological status Parity Last caretaker EEG Maternal smoking Last breastfeeding MRI Maternal drugs Who found the infant Neuropsychological development Type of delivery Where the infant was found Motor development Sex Co-bedding Metabolic parameters Birth weight Co-sleeping Infectious parameters Gestational age If parent was asleep Umbilical artery pH Blood pH and Base Excess Neurological symptoms Hypothermia treatment Hypoglycaemia Ventilation CPAP Intubation Brain Ultrasound EEG ECG Acute blood samples

4 RESULTS AND DISCUSSION

4.1 STUDY I AND II – THE BREATHING BRAINSTEM In the first part of this thesis, we demonstrated that the respiratory regions preBötC and pFRG/RTN maintain their functional organization and activity in organotypic cultures. Furthermore, we discovered a novel signaling pathway in central chemosensitivity involving

PGE2 and the EP3R.

The complexity of the respiration-related neural networks of the brainstem (see 1.1) contributes to the difficulties of creating an optimal in vitro model system (see 1.3). To provide sufficient generalizability, in vivo conditions should be well represented and provide sufficient details and manipulation of cellular and molecular mechanisms. To make the respiratory networks readily available for visualization, electrophysiology and manipulation over periods ranging from hours to weeks, we developed a new method where the preBötC and pFRG/RTN were maintained in organotypic brainstem slice cultures (study I). A similar system for mouse hippocampus has been used to investigate neural network organization previously (167, 265). However, hippocampal cultures are less endogenously active than respiratory networks. Therefore, the respiratory slice cultures might be more advantageous, not only for respiratory studies, but also for studying neural network activity and plasticity in general.

The organotypic slice cultures of the respiratory brainstem regions displayed intact neurons and the presence of Nk1R indicated cytoarchitectually preserved respiratory regions of the brainstem. Within the regions, we could also observe the distribution of glial cells, i.e. astrocytes and microglia (study II). The protein expression pattern was preserved during three weeks of cultivation (see Figure 4a), with the only morphological change observed being a thinning and widening of the slices. Few necrotic and apoptotic cells where found in the brainstem slice cultures, and only in the thicker regions relating to insufficient oxygenation locally (167), as observed by Gahwiler and colleagues (177). This effect limits the use of organotypic slice cultures in adolescent and adult animals. Slices have to expand over a certain range to be able to include large enough parts of the respiratory regions for rhythm generation (151, 179). This range increases with animal age, requiring thicker slices for proper rhythm generation. However, improvements on survival of thicker slices have been made using an interstitial microfluidic perfusion technique, allowing slices up to 700 µm thick to be kept alive for more than five days (266). Oxygen and glucose deprivation (267), caused an extensive necrosis (study II). Electrophysiological recordings showed well- functioning and spontaneously active neurons (study II). Overall, we can conclude that, at single cell level, both the preBötC and pFRG/RTN were well-preserved in the organotypic brainstem slice cultures for several weeks.

Utilizing Ca2+ time-lapse imaging, we studied the activity of multiple cells simultaneously. This revealed a synchronized network, with a small-world structure in both the preBötC and pFRG/RTN that was preserved for three weeks of cultivation (see Figure 4b, study II). Such

network topology of the preBötC has been described previously based on neuronal staining (268). Furthermore, this organization has been suggested to have evolutionary advantages, as it reduces the metabolic demands on the network and increases its robustness (269, 270). This because the small-world network structure allows specific nodes to have a specialized function within the network, while demands on the networks is distributed over several nodes. These properties does not only occur in biological system, but are also found in the overall organization of the Internet and social connections as those seen on Facebook (263).

Compared to the previous morphological studies, our analyses are based on investigating the functional activity of multiple cells of multiple types, including both neurons and glia, and thus provides a more complete picture (147, 271). We could also demonstrate that many of the cellular interactions in the preBötC are based upon gap junction coupling. Such coupling has been described to be important early in development (46) and gap junction-mediated Ca2+-transients provide a template for the proliferation of neural progenitor cells and development into more mature neural networks (146, 270). Furthermore, gap junctions are essential for maintaining the respiratory frequency (272). In our setting, neuronal Ca2+ signaling frequency of individual cells was unaffected by gap junction inhibition while most synchronization disappeared. This further emphasizes the importance of network synchronization and collaboration between cells.

However, during gap junction inhibition, a smaller connected network remained, suggesting the presence of a gap junction-independent, chemical synapse driven subnetwork in the preBötC. These results led to the hypothesis of the presence of separate neuronal and glial networks with different signaling systems (144, 271), further examined in study III. Gap junction inhibition did not affect pFRG/RTN synchronization. Taken together, we confirmed that the preBötC synchronization is dependent on gap junctions during the neonatal period (272, 273), while the pFRG/RTN is not (62).

From the brainstem slice culture, we recorded rhythmic respiratory-related output from the 12th cranial nerve and hypoglossal nucleus (study II). Activity was, just as the network synchronization, preserved for up to three weeks in culture. The motor output frequency from the brainstem slice cultures was within the previously described range for transverse medullary slices in vitro (165, 274). The output also responded with an increased frequency after Substance P administration and a decrease in frequency during µ-opioid receptor activation (see Figure 5). Thus we demonstrated that the breathing brainstem can be maintained in the organotypic brainstem slice cultures for up to three weeks. This expands the possibilities of detailed investigation of respiratory network over longer periods of time as well as during long-term exposure to exogenous factors. In several clinical conditions, the brain and brainstem are affected by intermittent exposure to e.g. hypoxia or hypercapnia over days to weeks (275-277). The exposure could also be continuous for hours to weeks, such as during systemic inflammation (120, 219).

Inflammation is related to respiratory dysfunction in neonates (see 1.5). Furthermore, the transfer of inflammation across the blood-brain barrier is a localized process and not

dependent on a generally increased PGE2 production in the brain (278). In study II, we hypothesized that PGE2 directly modifies respiratory network behavior. Indeed, we show that

PGE2 has a direct effect on both the preBötC and the pFRG/RTN, mediated via the EP3R. 2+ PGE2 increased the pFRG/RTN Ca signaling frequency, but inhibited the preBötC frequency. The PGE2 effect on the preBötC may be concentration dependent as higher concentrations have been shown to increase respiratory rhythm in the preBötC. This is mediated via an alteration of the calcium-activated non-selective cation conductance (ICAN)

(121). During neonatal infections, PGE2 is found at picomolar ranges in the cerebrospinal fluid (156). In study II, a nanomolar range was utilized to compensate for the in vitro 2+ conditions. On a single cell level, PGE2 increased width and amplitude of the Ca peaks in a manner dependent on INAP. This could mimic the PGE2 induction of sighs in vivo (study II) and in vitro (121). The opposite effect of the same molecule in the two different respiratory networks might depend on a different distribution of different subtypes of EP3Rs (see 1.5.1). The preBötC contained a larger fraction of inhibitory EP3Rs compared to pFRG/RTN, where the stimulatory subtype was most abundant.

In summary, these results provide evidence of how PGE2 and inflammation may cause respiratory depression and apneas during infectious periods in neonates (116, 156, 219, 279).

Furthermore, PGE2 levels are high at birth (280), which is an important physiological response for the transition to extrauterine life (281, 282), promoting deep breaths (121, 283) and relaxation of the airways (284). However, inflammation also disturbs the response to hypercapnia in neonates (116, 118, 152). Therefore, we investigated the hypercapnic response of the pFRG/RTN (62) and its relation to inflammation.

We could conclude that the hypercapnic response is gap junction dependent (study II). This corroborates recent findings on the CO2 sensing properties of Cx26 (203). Such Cx hemichannels are known to release ATP during hypercapnia, which stimulates breathing through actions on purinergic type 2 receptors (62, 147, 153). In vivo, the hypercapnic response leads to increased respiratory frequency and tidal volume, as well as sigh frequency

(study II), similar to the effect of PGE2. Therefore, we hypothesized that, in addition to ATP,

PGE2 is also released through gap junctions. Indeed, during hypercapnic challenge, the PGE2 concentration in the microenvironment of the brainstem slice culture transiently doubled.

Such a release of PGE2 was absent in all brainstem slice cultures treated with gap junction inhibitors. Furthermore, genetic ablation of Ptger3 reduced the hypercapnic response both in vivo and in vitro, similar to previous results (152). Moreover, pharmacological blockage of EP-receptors and optogenetic inhibition of Ptger3-expressing cells in the pFRG/RTN blunted the response CO2.

The discovery of the novel PGE2-EP3R pathway in the hypercapnic response links inflammation to respiratory control further. The reduced responsiveness to CO2 during inflammation and infections (116, 152) could be related to saturation or disruption of the signaling pathway. Furthermore, as PGE2 levels are high immediately after birth (280) the responsiveness of the brainstem could be reduced (see study IV, 4.3).

Figure 4. Respiratory networks in organotypic cultures. In the novel organotypic culture system of the respiratory brainstem, the cytoarchitecture remained intact. With immunohistochemistry, the expression of the neural markers Nk1R, Map2, Kcc2, Tuj1 and VGlut2 was evaluated (a). Utilizing Ca2+ imaging, the functional connections between individual cells was investigated. This visualized the network structure of both the preBötC and the pFRG/RTN. Both networks were organized in a small-world network structure. Colored lines represent the correlation coefficient between the analyzed cell pair. Warmer colors indicate a higher correlation coefficient. All nodes in the network represent the coordinates of a cell in the brainstem slice culture. Scale bars; 100 µm.

Figure 5. The breathing brainstem in a dish. Even after three weeks of cultivation, motor output could be recorded in the hypoglossal nucleus. This rhythm was regular under control conditions (top trace) and increased after application of Substance P (1 µM, middle trace) and decreased after application of µ-receptor agonist [D-Ala2, N-Me-Phe4, Gly5-ol]- enkephalin (DAMGO, 0.5 µM, bottom trace). This strongly indicates that respiration-related rhythmic motor output remains.

4.2 STUDY III – BREATHING BY GLUE Two major questions emerged from study II. First, do astrocytes play a role in the network organization of the respiratory regions? Second, are astrocytes the source of the PGE2 released during hypercapnic challenge? Since the PGE2 release was gap junction-dependent and mPGEs-1 was present in pFRG/RTN astrocytes, these cells were a likely candidate for the PGE2 production. Furthermore, previous studies had found that astrocytes release ATP during hypercapnia (153, 201). It is also known that astrocytes are involved in the CO2 response as astrocyte-specific knockout of MeCP2 blunts the CO2 response (204).

To investigate the role of astrocytes in the preBötC and pFRG/RTN, we bred a transgenic mouse line with GFP-labeled Gfap expressing cells (i.e. astrocytes (285)). In these mice, the astrocytes also express the MrgA1R, making it possible to specifically stimulate astrocytes. Through IHC, the expression of GFP was shown to overlap with Gfap and S100β, but not neuronal or microglial markers. This suggests an astrocyte-specific expression of GFP, even though we cannot fully exclude the presence of undifferentiated Gfap-expressing precursor cells (146).

Ca2+ time-lapse imaging data in study III revealed two subgroups of astrocytes, based on the presence of rhythmic Ca2+ oscillations. The majority of astrocytes were silent, while a smaller subset had a rhythmic Ca2+-signaling pattern. Such subgrouping of preBötC astrocytes have been suggested previously, based on both electrophysiological and Ca2+ imaging methods (141, 142, 145). However, no previous study has provided such data from pFRG/RTN. In study III, we did not measure hypoglossal motor activity and can therefore not evaluate if the astrocytic oscillations are phase-locked with the respiratory rhythm. At present, there is no strong evidence for phase-locking between astrocyte signaling and respiratory output. Schnell and colleagues did not find any relation between astrocyte oscillations and respiratory rhythm (141). Okada and Oku and colleagues suggested that astrocytic Ca2+ oscillations are pre- inspiratory (144, 145) and that they are correlated with respiration-related neuronal activity (145). However, it remains to be investigated if astrocytes can entrain the respiratory rhythm.

The active astrocytes were synchronized in their Ca2+ oscillations and organized in a network within the respiratory regions, different from the non-astrocyte (i.e. neuronal) network. Several connections between the two networks were found, similar to what has been shown previously (145). Interestingly, the astrocytic network constituted a larger proportion of the total network in pFRG/RTN compared to the astrocytic proportion of preBötC. The pFRG/RTN network also contained more astrocyte-neuron connections than the preBötC. The measured correlation does not imply causality and does not provide any detailed information on the signaling between the cells. However, the different proportions do suggest a different role of astrocytes in the two regions. This could be related to the role of astrocyte in chemosensitivity within the pFRG/RTN, which could require more substantial communication between the cell types (286).

The astrocytes displayed a low oscillating frequency, similar to that described previously for active astrocytes (141, 142, 144, 145). This frequency increased when the astrocytes were stimulated by the addition of the MrgA1R ligand FLRF. In the pFRG/RTN, neuronal Ca2+ oscillation frequency increased in response to astrocyte stimulation. This effect was absent in the preBötC. The network structure of neither regions was affected by astrocyte stimulation.

Although astrocytes did not seem to modify neuronal activity in the preBötC, we cannot exclude an important role in respiratory rhythm generation. For example, astrocyte inhibitor methionine sulfoximine depresses breathing in vivo (139), and glial inhibitors (fluorocitrate, fluoroacetate, and methionine sulfoximine) decrease preBötC activity in vitro (138, 140). Furthermore, the data in study III suggest that pFRG/RTN astrocytes do modulate respiratory network activity. This corroborates the results of previous studies that have demonstrated the involvement of astrocytes in chemosensitivity through purinergic signaling (147, 153, 201, 287, 288). Furthermore, in study II we showed that hypercapnic challenge triggers a release of PGE2. Therefore we hypothesized that astrocyte specific stimulation would do the same.

Indeed, we found that FLRF application caused a doubling of PGE2 concentration in the microenvironment of pFRG/RTN. This was not seen in brainstem slice cultures of preBötC nor in wild-type brainstem slice cultures. Furthermore, the hypercapnic response was reduced after activation of astrocytes, suggesting that PGE2, and probably other gliotransmitter, stores were depleted. This relates to the effect of inflammation on the hypercapnic response (116,

152). As EP3Rs are found on astrocytes (study II), PGE2 could exert an autocrine effect in the hypercapnic response to emphasize it. Exogenous PGE2 released from endothelial cells during systemic inflammation (156) could therefore activate pFRG/RTN astrocytes. This would deplete the gliotransmitter stores resulting in a blunted hypercapnic response as observed in study III. Furthermore, astrocytic activation by PGE2 could further explain the stimulatory effect of PGE2 in the pFRG/RTN (study II).

The mechanism behind the PGE2 release from astrocytes remains to be investigated. CO2 affects intracellular pH and drives bicarbonate and sodium ions into the cell. This triggers a 2+ Ca influx (134) and subsequently stimulates cell activity. In parallel, CO2 can directly modify Cx26 (201, 203). Thus, the experimental setup with FLRF-induced activation and Ca2+ influx cannot discern the specific details the gliotransmitter release during hypercapnic challenge.

4.3 STUDY IV – SUDDEN UNEXPECTED POSTNATAL COLLAPSE In the fourth and final study of this thesis, we identified 115 cases of Sudden Unexpected Postnatal Collapse (SUPC) among the total of 313 351 seemingly healthy infants born >35 weeks of gestational age. This amounts to an incidence of 36.7 per 100 000 live births, which is higher than previously reported (244, 245, 289-292). However, at present, no ICD-10 diagnosis exists for SUPC. This limits the comparability of studies performed. Evidently, overlapping terminology and a large variation of inclusion criteria among studies, results in a wide-spread incidence of SUPC, ranging from 2.6 to 133 cases per 100 000 live births (245, 293).

Compared to previous studies, study IV have several advantages. First, study IV is one of the largest studies to date. The majority of previous investigations concern case reports of a few patients and only includes collapses occurring during the first hours after birth (245, 294). Some regional and national studies with larger cohorts exist (291, 295-297). However, most of these studies rely on reporting of cases by hospital staff or parents (291, 294, 297, 298). Thus, they are at risk of reporting bias as well as incomplete reporting. The cases in study IV were identified through thorough investigation of medical records identified by related ICD- 10 diagnoses. This increases the reliability of the data and could explain why we have identified more cases compared to the previous investigations. We urge the need for a specific ICD-10 diagnosis with disease criteria similar to the inclusion criteria used in study IV. This could both increase awareness of the condition and improve the comparability of future research.

For comparison, we also investigated patients diagnosed with Group B Streptococci (GBS) sepsis. GBS sepsis is a feared, and known, disease in the clinics (299). We identified 68 cases, amounting to an incidence of 21.7/100 000 live births. During the study period, the incidence of both SUPC and GBS sepsis have declined, although the SUPC incidence constantly have been higher (Figure 6). This reduced incidence could be related to an increased awareness and use of the national guidelines that exist for GBS sepsis (300) and was composed for SUPC during this time (301).

All included babies in study IV were seemingly healthy and showed no signs of asphyxia during birth. The average Apgar scores were 8.4 ± 1.4 at 1 min, 9.7 ± 0.7 at 5 min and 9.8 ± 0.4 at 10 min. 0.9 % of the SUPC cases had an Apgar score of <7 at 5 min, which is similar to regional (Stockholm County) and national (Sweden) data (1.0 % and 1.2 % respectively) but lower than babies diagnosed with GBS sepsis (2.9%). Umbilical artery pH at birth was 7.24 ± 0.08, which is not different from healthy babies; 7.25 ± 0.07 (302). Considering parity with the mother, SUPC cases has a higher proportion primiparous mothers compared to both regional and national data (66% vs 47 % and 45 %). The proportions of different types of delivery (vaginal or caesarian section) was not different between SUPC cases and regional or national data. Thus, SUPC is not related to adversities around birth. However, we found that SUPC cases had a lower birth weight and were born at an earlier gestational age compared to the regional and national data. We did not examined the pregnancy period and can thus not exclude that there are identifiable risk factors presenting earlier.

The majority of SUPC cases occurred within 24 hours after birth (83 %) and half (52 %) within the first 4 hours (see Figure 7a). This explains why most collapses occurred in either the delivery room (44 %) or the maternity ward (44 %). Despite occurring in the hospital, 87 % of the cases collapsed while in the parents’ care. Only 3 % (n = 4 infants) collapsed at home, but 3 out of these 4 died. An additional 5 SUPC cases died following the collapse. Of these eight, 7 collapsed within 24 hours. Five of the 8 dead were treated with hypothermia. An additional eight patients were hypothermia treated after the collapse, but all except one developed neurological sequela such as hypoxic ischemic encephalopathy (HIE). In total, 25

% (n = 28) suffered from neurological sequela. Among these 25 % one third developed HIE grade I (HIE I), one third developed HIE II. One SUPC case resulted in HIE III and that infant later died.

In the total cohort, 70 % of the SUPC cases were associated with co-bedding (sleeping in the same bed as the caretaker) similar to SIDS (303) and all infants who died collapsed during co-bedding with a parent. Thus, the beneficial Skin-to-Skin care needs to be assessed with caution (246, 304, 305). Guidelines to reduce SUPC and SIDS incidence exist in Sweden (301), the United Kingdom (306) and the United States (305). These emphasizes the importance maintaining focus on the infant during the first hours after birth. Safe skin-to-skin care includes keeping the infants airways clear in a stable position while hospital staff regularly monitors the mother with her infant. Furthermore, when the mother wants to sleep, the infant must be placed in a bassinet or a support person who is awake and alert (305). National guidelines for the prevention and treatment of GBS sepsis (300, 307) have minimized the incidence (308) and reduced mortality to zero (study IV). Implementation of the existing guidelines for SUPC both nationally and internationally could similarly improve the care and reduce mortality of infants.

As part of the prospective arm of the study, urine samples were taken in 8 of the SUPC cases and an additional 12 age-matched control infants. The urine was analyzed for a PGE2 metabolite (U-PGE2m). The levels of this metabolite was high immediately after birth, confirming previous studies (280) and remained elevated the first 3 days postnatally (see

Figure 7b). We saw no difference between the SUPC cases and control in terms of U-PGE2m levels or dynamics. As the period of increased PGE2 levels coincide with the period when collapses occur, PGE2 could be of pathogenetic importance in SUPC.

Figure 6. The SUPC incidence is higher than the GBS sepsis incidence. Throughout the study period of study IV, the incidence of SUPC has been higher than the GBS sepsis incidence. Still SUPC is relatively unknown among hospital staff and the condition is inadequately studied compared to the feared and well-known GBS sepsis. However, both incidences are declining, which could depend on the establishment of national guidelines and increased awareness of the conditions.

Figure 7. The first 24 hours after birth are critical. The majority (83 %) of SUPC cases occurred within 24 hours after birth, and half within the first 4 hours (a). This correlates with the same time when PGE2 levels are high as a consequence of the stress of being born (b).

We hypothesize that the increased PGE2 levels limits the infant’s ability to adjust its breathing adequately when pCO2 rises.

5 CONCLUSIONS This thesis shows that organotypic cultures of transverse medullary slices can maintain respiratory-related networks and output for several weeks in vitro. It expands the possibilities for long-term experiments including viral transfection and drug applications. Using this novel system, we report that astrocytes are involved in the basal brainstem respiratory network activity. Within the preBötC and pFRG/RTN there are subgroups of astrocytes that form their own individual cellular network, which is interconnected with the neuronal network. Stimulation of the astrocytes does not affect the preBötC neurons, but instead increases pFRG/RTN neuronal activity, similarly to hypercapnia. During hypercapnia and astrocyte stimulation, PGE2 is released. PGE2 then directly modifies respiratory networks through its actions on EP3R (see Figure 8). Elevated PGE2 levels blunts the central pattern generators’ response to hypercapnic events. PGE2 levels are increased at birth. Thus, an infant’s ability to adjust its breathing and arouse when pCO2 increase is limited. This could contribute to Sudden Unexpected Postnatal Collapse and death. In summary, this thesis provide novel evidence of mechanisms behind the vulnerable infant in the triple-risk model for SIDS. We also identified the importance of the first hours after birth. Implications of guidelines for the care during this period could reduce the risk of both neurological sequela and death. Furthermore, the inflammatory pathways and astrocytes are new therapeutic targets for refining methods to detect infants at risk of developing autonomic failure.

Figure 8. Model of how PGE2 modulates respiration through actions on the brainstem central pattern generators. Systemic inflammation, through IL-1β, and hypoxia, induces the production of PGE2 in blood-brain barrier (BBB) endothelial cells (156). PGE2 subsequently induces respiratory depression and increases sigh activity via the inhibitory G-protein coupled receptor EP3Rα in the preBötC. In the pFRG/RTN, PGE2 plays a role in the response to elevated pCO2. CO2 directly modulates Cx26 hemichannels, leading to ATP release. Cx26 also releases PGE2 from astrocytes. PGE2 then increases respiratory activity via the stimulatory G-protein coupled receptor EP3Rγ on pFRG/RTN neurons. Thus, inflammation, hypoxia, and hypercapnia alter respiratory neural network and motor output and breathing activity through distinct effects of PGE2 in the pFRG/RTN and the preBötC, respectively. Elevated PGE2 levels, as observed during ongoing inflammation and at birth, may decrease the central pattern generators’ ability to respond to hypoxic and hypercapnic events. Therefore, an infant’s ability to adjust its breathing and arouse when pCO2 levels increase is limited. This could lead to SUPC and may have fatal consequences.

6 FUTURE PERSPECTIVES The results presented in this thesis have generated several interesting and important questions for future research and possibilities to better the care of infants:

 How does the role of astrocytes in respiratory rhythm generation change during development and is this related to the risk of respiratory dysfunction?

 Would modifying the PGE2 pathway have benefits in the treatment of apnea of prematurity?  How shall safe skin-to-skin care be implemented to not increase the risk of SUPC and SIDS? What is the effect of existing guidelines?  Could changes in respiratory activity be used as physiomarkers to identify life- threatening events and infectious episodes early in preterm and term infants?

7 ACKNOWLEDGEMENTS I want to acknowledge the mice that unknowingly and unwillingly have had their lives sacrificed for this research. Without them and their ultimate sacrifice these discoveries could not have been made and research would be at a standstill. We should all remember non-human animal’s contribution to science. Several people have contributed to this thesis and supported me through the work. However, quite a few deserve special thanks. My main supervisor Eric Herlenius, who took a chance by first letting me join the lab even as I had no previous experience. Even more courageous of him to give me my own project early on. You have helped me develop into an independent researcher and managed to guide me through the scientific work. This have led to several great accomplishments in both research and life. Your never-ending enthusiasm amazes me. A true “eldsjäl” in science. My co-supervisor Per Uhlén, for necessary technical assistance and being a great scientific role model. Sri, for teaching me the essential knowledge that later became the fundament for this whole thesis. Your strictness in scientific methodology and laboratory work really offered me a great starting position for my future experiments. Zachi Horn, who supported and assisted me in all the scientific work. You are probably the one who have taught me the most, both when it comes to biomedical techniques and being a PhD student. Evangelia Tserga, who did most of the weekend and evening experiments and always helped out when help was needed. I wish you all the best in your future endevours. Thomas Ringstedt, for all the work and help with study III of this thesis and who has played a significant role in scientific discussions and adventures of both mind and body outside the laboratory. Lars Björk, I have learned so much from you, from basic scientific theory to detailed microscopy, and you made the work a lot more fun! Thomas Liebmann, Erik Smedler, Linda Westin, Martin Becker and Gintare Lasaviciute for unselfishly spending your time to help me out and solve my problems. Veronika Siljehav for all the advice on the research performed in the lab and on how to survive during a PhD. Also, for being a reminder that there are actual patients out there. Torkel Mattessson, for all the assistance, inspiration and a great friendship. You have increased my self-esteem in both life and science. I hope to collaborate with you in the future. Antoine Honoré, your expertise saved me weeks in just the last year. Where were you from the start? Also, many thanks for babysitting when needed and teaching Love the basics of computer programming. Cecilia Dominguez for managing the lab like a boss. Jennifer Frithiof for simply fixing what needed to be fixed. And for times of ventilating thoughts and frustration. Ruth Detlofsson, for being a good friend, always providing assistance and taking care of the lab when no-one else did. Yuri Shvarev, for supporting and questioning my research, making it better.

Gilad Silberberg, for introducing me to electrophysiology and being a real inspiration for high- quality research. Marie Carlén and Ed Boyden, for providing the tools for and all the help with optogenetics. Cici Dyberg, for widening my research field of view and keeping me in shape. Jelena Milosevic, for introducing me to the research education committee. Louise Steinhof, for the collaboration in study IV and providing a introducing a different side of medicine. Elin Doyle and Camilla Helmersson for helping out and giving me the possibility to teach and supervise. This has not only increased my scientific knowledge but also improved my skills in understanding and communicating research. Wiktor Phillips, Hanna Ingelman-Sundberg, Shanie Saghafian Hedengren, Anna Nilsson and Emilja Wilsson for providing an outstanding discussion platform in all aspects of science. Jan Philipp Reising and Amaia Nazabal, for help with lab work, fun discussions and great input to this thesis. My colleagues at the lab, Kerstin Jost, Birgitta Böhm, Elena DiMartino, Ahmed Osman, Wei Han, Kai Zhou, Iga Dobkowska, Makiko Oshima, Giulia Zanni, Monique Havermans and Lea Ballenberger for keeping work interesting, fun and challenging. Carin Widén, who introduced me to pediatrics, neonatology and boosted the interest that generated this thesis. Hugo Lagercrantz, who has been an inspiration from before my PhD and for providing the foundation on which this thesis rests. Henrik Källberg for being a great travel companion and discussion partner. My friend David Lagman, who has been my mentor in science. You have always supported my work and provided valuable inputs to all its parts. I’ve had great times dwelling in scientific know- how and discovering short-comings in other’s work. My friend Jon Samuelsson, who made the path towards academia both easier and more fun. Without some, I would probably not have been here today. Leo Johansson, for all the assistance at work, ranging from technical support to custom building anesthesia units, and for great fun outside work. Sabine and Kjell Noresson, for inspiration, relaxation and practical help during times of stress and big workload. Gunnel Forssberg for helping out outside work, liberating time for research. My brother Simon, for supporting me and feeding the scientific interest. Mom and Dad, for everything that has led to this. For teaching me how to be sensible, logical, responsible and practical. Optimus “Grisen” Prime and Tiszla for being great companions in life. Love, for all the joyfulness, motivation, and inspiration. Josephine, for all the love and support. Always reminding me of the important things in life.

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