ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2018

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1472

Evolution of acetylcholinereceptors and study of theanatomy of the mouse brainreward system

JULIA E. PEDERSEN

ISSN 1651-6206ISBN 978-91-513-0366-6urn:nbn:se:uu:diva-353989

Dissertation presented at Uppsala University to be publicly examined in Biomedicinsktcentrum C4:301, Husargatan 3, Uppsala, Thursday, 6 September 2018 at 09:15 for thedegree of Doctor of Philosophy (Faculty of Medicine). The examination will be conductedin English. Faculty examiner: PhD Arnaud Chatonnet (Department of Animal Physiology andLivestock Systems, French National Institute for Agricultural Research, Montpeiller France).

AbstractPedersen, J. E. 2018. Evolution of acetylcholine receptors and study of the anatomy of themouse brain reward system. Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1472. 54 pp. Uppsala: Acta Universitatis Upsaliensis.ISBN 978-91-513-0366-6.

This thesis work is divided in two parts. In the first part, I make use of the transgenic TRPV1-Cre mouse line as a tool to investigate the midbrain ventral tegmental area (VTA). By using aChR2-EYFP construct, detailed mapping of connectivity shows that TRPV1-Cre VTA neuronsinnervate many brain areas such as the prefrontal cortex (PFC), ventral pallidum, bed nucleus ofstria terminalis and lateral habenula. Interestingly, a mainly excitatory subcircuit from the VTAto PFC in the TRPV1-Cre mouse was identified which suggests a fast modulatory mechanismof the PFC by a VTA subpopulation. These results are discussed in the light of behavioral andneurophysiological literature. In the second part, the evolution of the vertebrate acetylcholine(ACh) receptor gene families in relation to the whole genome duplications (WGDs), also called1R and 2R, was investigated. The nicotinic ACh receptors (nAChRs) form a complex genefamily, where the members have evolved with varying rates. Our analyses combined phylogeny,intron positions and chromosomal synteny in order to elucidate the nAChR evolution in relationto the vertebrate WGDs. We found that ten ancestral nAChR genes were present prior to theWGDs. 1R and 2R then expanded this set to 19 genes, of which 16 are present in mammalstoday. The teleost specific WGD, 3R, further expanded the repertoire into 31 genes, of which27 genes are present in zebrafish. The muscarinic ACh receptors (mAChRs) on the other handform a smaller receptor family. Using the same approach, our analyses show that there were twoancestral genes present prior to the WGDs, expanding to five genes following 1R and 2R. Inzebrafish, all genes retained duplicates in 3R resulting in ten mAChR genes present today. Ouranalyses also showed that four mAChR teleost genes have gained introns, some up to six introns.The evolutionary analyses of the receptor gene families show that all vertebrate duplicationevents in the AChR families, except for two among the nAChR genes, occurred through 1R, 2Rand 3R, displaying the substantial impact of the WGDs on the evolution of the AChR genes.

Keywords: Ventral Tegmental Area, Mesocorticolimbic system, Glutamate, Optogenetics,Histology, Acetylcholine, Receptor, Muscarininc, Nicotinic, G protein-coupled receptor, Geneduplication, Tetraploidization, Synteny, Paralogon, Ohnolog, Zebrafish, Spotted gar

Julia E. Pedersen, Department of Neuroscience, Pharmacology, Box 593, Uppsala University,SE-75124 Uppsala, Sweden.

© Julia E. Pedersen 2018

ISSN 1651-6206ISBN 978-91-513-0366-6urn:nbn:se:uu:diva-353989 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-353989)

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Pedersen, J.E. The connectivity of the TRPV1-Cre mouse line

as a useful tool for exploring the function of a VTA subpopula-tion. Manuscript.

II Pedersen J.E., Bergqvist C.A., Larhammar D. Evolution of ver-tebrate nicotinic acetylcholine receptors. Manuscript.

III Pedersen J.E.*, Bergqvist C.A.*, Larhammar D. Evolution of the muscarinic acetylcholine receptors in vertebrates. Manu-script.

* Authors contributed equally.

Contents

Introduction ..................................................................................................... 9 How to make a brain .................................................................................. 9 Part I: The brain reward system ............................................................... 10 

Brain structure and function ................................................................ 10 The mesocorticolimbic system ............................................................ 12 Behaviors related to VTA circuit activity ............................................ 14 Novel VTA subcircuitries arising from the TRPV1-Cre VTA subpopulation ....................................................................................... 15 

Part II: Evolution of receptor gene families in vertebrates ....................... 15 Vertebrate genome evolution ............................................................... 15 The nicotinic acetylcholine receptors .................................................. 20 The muscarinic acetylcholine receptors ............................................... 23 The evolution of the ACh receptor gene families ................................ 24 

Aims .............................................................................................................. 26 Specific aims for each study ..................................................................... 26 

Experimental procedures .............................................................................. 27 Part I ......................................................................................................... 27 

Animals and ethical considerations ..................................................... 27 Optogenetics – virus injection and light stimulation ........................... 27 Immunohistochemical staining procedures ......................................... 27 

Part II ........................................................................................................ 29 Amino acid sequence retrieval and multiple sequence alignment ....... 29 Phylogenetic analyses .......................................................................... 29 Conserved synteny and paralogon analysis of neighboring gene regions ................................................................................................. 29 Intron position analysis ........................................................................ 30 

Results ........................................................................................................... 31 Paper I ...................................................................................................... 31 Paper II ..................................................................................................... 32 Paper III .................................................................................................... 34 

Discussion ..................................................................................................... 36 The VTATRPV1-Cre population as a model for investigating VTA function (Paper I) ..................................................................................... 36 The evolution of the nAChR gene family and its expansion in the vertebrate WGDs (Paper II) ..................................................................... 37 All vertebrate mAChR genes originate from the vertebrate WGDs (Paper III) ................................................................................................. 39 The evolution of receptor gene families (Paper II and III) ....................... 40 

Conclusions ................................................................................................... 41 

Future perspectives ....................................................................................... 43 

Acknowledgement ........................................................................................ 45 

References ..................................................................................................... 46 

Abbreviations

ACh, acetylcholine AH, anterior hypothalamus Ahi, amygdalohippocampal area aLRT SH, approximate likelihood ratio test Shimodaira–Hasegawa AORe, ancestral ohnologs resolution BD, binding domain BNST, bed nucleus of stria terminalis CHRN, cholinergic receptor nicotinic CHRM, cholinergic receptor muscarinic ChR2, channelrhodopsin-2 CLi, caudal linear nucleus Co, cortical amygdala DAPI, 4',6-diamidino-2-phenylindole DAT, dopamine transporter DR, dorsal raphe ECD, extracellular domain EL, extracellular loop EYFP, enhanced yellow fluorescent protein GAD, glutamic acid decarboxylase GPCR, G protein-coupled receptor HTR3, 5-hydroxytryptamine receptor 3 ICD, intracellular domain IF, interfascicular nucleus IL, intracellular loop KO, knock out LHb, lateral habenula LGIC, ligand-gated ion-channel LORe, lineage-specific ohnologs resolution LS, lateral septum

mAChR, muscarinic acetylcholine re-ceptor ML, maximum likelihood MnR, median raphe nucleus Mya, million years ago NAc, nucleus accumbens NAcC, nucleus accumbens core nAChR, nicotinic acetylcholine re-ceptor NAcSh, nucleus accumbens shell NMJ, neuromuscular junction OMIM, online mendelian inheritanceof man PBP, parabrachial pigmented nuclei PFA, paraformaldehyde PFC, prefrontal cortex PhyML, phylogenetic maximum like-lihood PIF, parainterfascicular nucleus PO, preoptic area PN, paranigral nuclei RLi, rostral linear nucleus SN, substantia nigra SUM, supramammillary nucleus TH, tyrosine hydroxylase TM, transmembrane domain TRPV1, transient receptor potential cation channel subfamily V member 1Tu, olfactory tubercle VMH, ventromedial hypothalamus VGLUT2, vesicular glutamate trans-porter 2 VP, ventral pallidum VTA, ventral tegmental area

VTAR, ventral tegmental area rostralpart WGD, whole genome duplication ZAC, zinc-activated ion channel

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Introduction

Neuroscience is the study of the structure and function of the nervous system. It is a multidisciplinary research field that includes evolution and develop-ment, cellular and molecular biology, anatomy and physiology, pharmacol-ogy, behavior and cognitive psychology. Combination of disciplines is re-quired in order to study the complexity of the nervous system. Compared to other sciences such as physics, neuroscience is a biological science in which causality, i.e. the relationship between cause and effect, is not as straightfor-wardly established. That in turn puts high demands on the questions asked and methods used. By studying why organisms’ behaviors have evolved by for instance identifying driving forces of evolution, how the nervous system has come to be organized in the way it is, how it is built regarding anatomical, molecular and cellular maps, what behavioral outputs certain types of neu-ronal activity lead to and how this is shaped during development by for in-stance genetic and environmental impact, we gain understanding of the mul-tiple levels of causality that make a brain.

How to make a brain Ernst Mayr referred to immediate causes as the type of process that explains how internal processes (e.g. a specific form of brain activity or anatomical structure) and external factors (e.g. food or space availability) result in biolog-ical functions. Further, Mayr referred to ultimate causes as evolutionary ex-planations for why organisms have acquired specific traits (Laland et al., 2011; Mayr, 1961). The first part of this thesis addresses the first type of ques-tion; how may a specific form of connectivity between brain areas support specific forms of behaviors (reward and cognitive associated behaviors)? I studied the anatomy and connectivity of a subpopulation of neurons in the ventral tegmental area (VTA), part of the brain reward system, marked by the transgenic TRPV1-Cre mouse line (Lagerström et al., 2010; Viereckel et al., 2016), in order to investigate this specific marker as a model for increasing our understanding of the reward system. Then, during the second part of this thesis, the other type of question is addressed; why does such a structure end up carrying out such function? The "why" question is the type of problem that we could address for any trait or biological component that we think is rele-vant for the evolution of the brain. A classical approach before the emergence

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of the genomic era was comparative anatomy. However, anatomical compar-isons might be insufficient if biologists are interested in understanding the role of specific DNA sequences, or genes, in brain novelties across very distant species. In this context, whole genome duplications (WGDs) have been sug-gested to be important evolutionary events, potentiating species diversity. Therefore, I investigated the evolution of the acetylcholine (ACh) receptor gene families in relation to the vertebrate WGDs1. Although the evolutionary history of the receptor genes should not be conflated with their utility (see “Vertebrate genome evolution” section below, referring to Stephen Jay Gould's work on exaptation (1991, 2002)), or function, history is a first step into understanding why these genes are the way they are rather than some other way.

Part I: The brain reward system Brain structure and function In 1848, the railroad worker Phineas Gage got an iron rod crossing through his skull. He survived, but a large part of his left temporal lobe was severely damaged. Following the accident, he suffered personality and behavioral changes, and his friends did not any longer recognize him as the person he once used to be. Phineas Gage is one of the first documented examples of how lesion to a specific part of the brain is directly correlated with behavioral al-terations (Damasio et al., 1994). However, although lesion studies among other functional experiments have enabled us to relate some regions of the brain to certain types of behaviors, one-to-one relationships between a specific brain structure and function cannot be supposed, as brain regions do not act in isolation but are dependent and acting in parallel with other brain regions. To put it even simpler, the brain is not like the rest of the body, where fairly clear structure function relationships can be ascribed to separate organs. This is not how the central nervous system is organized and functioning, as it does not consist of separate entities working in isolation.

Before continuing with how to study and approach questions in brain struc-ture and function, we first need to have a look at how a brain area is defined. Korbinian Brodmann was one of the first to characterize and divide the ana-tomical structure of the cortex into different areas. His characterization was

1 A logical outcome of my neuro-functional studies could have been to investigate the evolution of the TRPV1 gene. However, as the TRPV1-Cre mouse line was used purely due to its potential as a marker of a subpopulation of neurons in the VTA, the TRPV1 gene in itself was not of main interest in this work. It appears that the TRPV1-CretdTom neurons in the VTA are not all express-ing TRPV1 and not all VTA TRPV1 neurons are expressing the CretdTom, in addition TRPV1 is mainly expressed during development and present at very low levels in the adult mouse (Vi-ereckel et al., 2016), thus evolutionary studies of the TRPV1 gene was not conducted. Instead, a complete evolutionary analysis of the ACh receptor gene families was conducted.

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based on cytoarchitecture, i.e. the size of the cell bodies, the shape, density etc. However, the borders determined by cytoarchitecture do not say much about the actual function. In fact, the borders of certain brain areas have been under discussion since the time of their description. An example of this com-plexity is the bed nucleus of stria terminalis (BNST), located close to the amygdala. The BNST was described already a century ago by Johnston J. B. (1923), but then it remained forgotten for half a century until it was brought up again by De Olmos and Ingram (1972). Until now, there has been a long debate on how to define the amygdala and the BNST, also referred to as the “extended amygdala”; should the extended amygdala perhaps even include the nucleus accumbens (NAc) (Swanson and Petrovich, 1998; Zahm, 1998)? One suggestions is that the NAc shell (NAcSh) acts as a transition area be-tween the striatopallidal system and the extended amygdala (Di Chiara et al., 1999; Heimer et al., 1991). This subject of controversy, i.e. how to define the amygdala and BNST, displays that anatomical characterization is not evident. In fact, as Swanson states in a paper from 2000; “The profound question really is not ‘what is the brain?’ but rather ‘what are the basic parts of the nervous system and how are they interconnected functionally?’” (Swanson, 2000).

Classically, functional studies have attempted to divide brain areas into ei-ther emotional or cognitive. Structures defined as emotional are for instance the regions in the limbic system, whereas classic cognitive areas have been exemplified by the cortex and hippocampus. Studies have indeed linked lim-bic structures, such as the VTA, to emotions (the VTA to NAc circuit for in-stance). However, the VTA is also linked to cognition (the VTA to prefrontal cortex (PFC) circuit). This is one of the reasons stated by Pessoa (2008) for why we cannot define brain regions as emotional or cognitive; many regions are both. Instead of attempting to apply one-to-one relationships between brain structure and function, what is found in the brain is many-to-many rela-tionships (Pessoa, 2008). As presented in a subsequent paper, Pessoa (2014) argues that in order to approach the relationship between brain structure and function and a many-to-many relationship, a network approach is required, i.e. scientists should no longer study separate, isolated parts of the nervous sys-tem. However, despite the evident theoretical complexity, methodologies im-pose limitations. We are still guided by and partly restricted to the classical methods to reveal structure-function relationships by mapping them one by one, and based on that try to understand the dynamics of the entire system. In the first part of this thesis, I worked according to this approach by using ge-netic and optogenetic methods in transgenic mice to study the characteristics of a specific subpopulation of neurons. Nevertheless, one needs to be careful when formulating the questions not to fall into the caveats just stated. Con-necting something as structurally complex as a brain with mind and behaviour

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is not straightforward2. An interesting example of the relationship between emotion and cognition is the connectivity between the VTA and the PFC. In paper I, I present functional and anatomical evidence supporting the role of a subpopulation of VTA neurons, marked in the TRPV1-Cre line, which may modulate the PFC by glutamatergic signaling.

The mesocorticolimbic system The mesocorticolimbic system forms a complex pathway, and as the name indicates it includes some major regions: the mesencephalon, cortex and lim-bic system. A central structure in this pathway is the VTA, located in the ven-tromedial part of the midbrain. The VTA efferents target for instance the NAc and PFC, hence the name of the pathway. The VTA in itself is a complex structure that consists of several subnuclei, namely the parabrachial pigmented nuclei (PBP), the paranigral nuclei (PN), the interfascicular nucleus (IF), the caudal linear nucleus (CLi) and the rostral linear nucleus (RLi) of the raphe, the parainterfascicular nucleus (PIF) and ventral tegmental area rostral part (VTAR) (Fu et al., 2012; Swanson, 1982). The VTA was classically defined as a homogeneous dopaminergic region, characterized by the expression of tyrosine hydroxylase (TH). However, it is now well established that this re-gion is not a homogenous dopaminergic region, but a rather heterogeneous area. In fact, it was shown more than 30 years ago that the VTA is not an entirely dopaminergic structure, as Swanson (1982) found that one third of the VTA neurons were TH-negative. A more detailed view was provided by Nair-Roberts et al. (2008), who showed that 35% of the neurons in the VTA were stained positive for the GABAergic marker glutamic acid decarboxylase (GAD) and 2-3% were stained positive for the glutamatergic marker the ve-sicular glutamate transporter 2 (VGLUT2). Further, it is now known that the VTA contains co-transmitting neurons, capable of dual-signaling with gluta-mate-dopamine (Yamaguchi et al., 2007, 2015; Zhang et al., 2015), glutamate-GABA (Root et al., 2014) or GABA-dopamine (Tritsch et al., 2012).

Detailed anatomical mapping based on molecular markers has further dis-played regional heterogeneity maps regarding the distribution of the marker. For instance, the medial VTA contains less TH than the lateral VTA (Yama-guchi et al., 2007, 2011), whereas opposite gradients are seen for VGLUT2

2 The more methods one combines, the more rigorous one should be in measuring and control-ling the variability of the components used. For example, optogenetic methods make use of viral strains that enter the cell depending on the properties of the specific serotype of virus, the type of tissue it enters etc. It is therefore crucial for the experimenter to know how each serotype works under each experimental condition, as different serotypes may affect the tissue differ-ently, especially in relation to the period of time during which the experiment is conducted (Jackman et al., 2014; Miyashita et al., 2013). Careful characterization of the transgenic line used is also of uttermost importance, in order to know the class of neurons and the circuit stud-ied (Lammel et al., 2015). Both of these factors are necessary for accurate interpretations of the results.

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(Yamaguchi et al., 2011). The GABA class of neurons on the other hand are distributed quite equally along the axes, but with differences between some subnuclei (Nair-Roberts et al., 2008). The VTA neurons establish local con-nections (Cameron and Williams, 1993; Dobi et al., 2010; Johnson and North, 1992), but also long range projections. Mapping of the regions targeted by the VTA efferents have shown that, in addition to the already mentioned PFC and NAc, which can be divided into the NAcSh and NAc core (NAcC), the VTA efferents also project to e.g. the olfactory tubercle (Tu), ventral pallidum (VP), bed nucleus of stria terminalis (BNST), lateral habenula (LHb) and amygdala (Figure 1) (Björklund and Dunnett, 2007; Ikemoto, 2007, 2010; Lammel et al., 2011; Morales and Root, 2014; Taylor et al., 2014), creating a highly in-terconnected network.

Figure 1. Schematic figure displaying central VTA efferent targets in the mesocorti-colimbic system. Abbreviations: PFC, prefrontal cortex; LHb, lateral habenula; LS, lateral septum; NAc, nucleus accumbens; VP, ventral pallidum; Tu, olfactory tuber-cle; Amyg, amygdala; LHb, lateral habenula; VTA, ventral tegmental area.

Studies have also investigated which type of VTA neurons, i.e. dopaminergic, glutamatergic or GABAergic, that are projecting to the respective target areas, to find that several target regions are innervated by at least two or sometimes all three VTA neuronal classes, for instance the PFC, NAc, VP, LHb and BNST (Björklund and Dunnett, 2007; Creed et al., 2014; Ikemoto, 2007, 2010; Lammel et al., 2011; Morales and Root, 2014; Taylor et al., 2014). In addition, studies of afferents and the areas projecting to the VTA have shown that all three VTA neuronal classes are targeted by the VP, LH and the dorsal raphe (DR) projections (Faget et al., 2016), of which the DR provides the single largest input, consisting of a mix of serotonergic, glutamatergic and GABAer-gic projections (Faget et al., 2016; Gocho et al., 2013). Differences in VTA afferent patterns have been reported (Beier et al., 2015; Faget et al., 2016; Watabe-Uchida et al., 2012), although some findings have been conflicting which is most likely linked to slightly different methodological approaches regarding for instance viral strains and transgenic mouse lines (Beier et al., 2015; Faget et al., 2016)..

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Behaviors related to VTA circuit activity In the 1950’s, Olds and Milner (1954) discovered that rats self-stimulated electricity into certain brain areas along the midline and they could henceforth distinguish rewarding, or reinforcing, areas in the brain from those mediating punishment (Olds, 1958; Olds and Milner, 1954). Since the time of these clas-sical experiments, reward and aversion associated brain regions have been mapped in greater detail. Olds and Milner showed that different brain regions could be associated with reward or aversion. Today it is also known that the regions associated with reward and aversion can overlap, as seen in the VTA in which some subcircuitries associate with reward, and some with aversion (Figure 2) (Berridge, 2007; Hennigan et al., 2015; Morales and Margolis, 2017; Tsai et al., 2009).

Figure 2. Schematic summary of VTA subcircuits and reward- or aversion associ-ated behavioral outcomes, depending on VTA neuronal class and connectivity; (a) displays glutamatergic input onto dopamine VTA neurons and their efferent targets, (b-c) displays glutamatergic, GABAergic or dopamine-GABAergic input onto non-dopamine VTA neurons and their dopaminergic or non-dopaminergic effects on ef-ferent target areas. Figure is reused with permission from Morales and Margolis (2017). Abbreviations: LHb, lateral habenula; VTA, ventral tegmental area; mPFC, medial prefrontal cortex; LDTg, laterodorsal tegmentum nucleus; nAcc, nucleus ac-cumbens; DRN, dorsal raphe nucleus; LHT, lateral hypothalamus; BNST, bed nu-cleus of stria terminalis; PV, parvalbumin; MSN, medium spiny neuron; VGLUT, vesicular glutamate transporter; VGAT, vesicular GABA transporter

Hence, the high degree of anatomical complexity in the VTA enables the or-chestration of a broad range of behaviors. Apart from reward and aversion, the VTA is linked to depression (Tye et al., 2013), fear (Abraham et al., 2014) and locomotion (Graybiel et al., 1994).

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Novel VTA subcircuitries arising from the TRPV1-Cre VTA subpopulation The TRPV1-Cre transgenic mouse line, where the Cre-expression is driven by the transient receptor potential cation channel subfamily V member 1 (TRPV1) promoter (Lagerström et al., 2010) was recently shown to mark a specific sub-population of VTA neurons (Viereckel et al., 2016). The neuronal classes marked in this VTA subpopulation included glutamatergic (62%), dopamin-ergic (7%) and GABAergic (23%) cells (Viereckel et al., 2016). The endoge-nous expression of TRPV1 is not fully mimicked in this Cre-line as 73% of the TRPV1 positive neurons express tdTom and 36% of the tdTom neurons ex-press TRPV1 in the P3 mouse and in the adult mouse the level of TRPV1 ex-pression is low (Viereckel et al., 2016). However, independently of the role of TRPV1, the TRPV1-Cre line provides an interesting and useful tool for in-creasing our understating of the VTA connectivity and function.

Part II: Evolution of receptor gene families in vertebrates Vertebrate genome evolution Most major animal phyla appeared as an explosion in the Cambrian radiation approximately 540 million years ago (Mya) (Erwin et al., 2011). Scientists have tried to understand which factors enabled speciation events of this mag-nitude, but as in most situations in biological science, it is difficult to rule out one component as the major driving force. For instance, there has been a de-bate regarding the contribution of increased oxygen levels (oxygen is required for the oxidative based metabolism required for e.g. the nervous system and muscles in animals) as a major driving force of the Cambrian explosion (Fox, 2016). It seems that the relationship between increased oxygen levels and the Cambrian radiation are not straightforward, and studies have suggested that the increase in oxygen levels at the time of the Cambrian radiation was actu-ally not as high as previously thought (Sperling et al., 2015), whereas others have suggested that fluctuations could have led to the levels necessary for ra-diation (Sahoo et al., 2016). Rather, there seems to be a combination of factors such as environmental, genetic and developmental that together enabled the passing of a threshold and a massive radiation to occur (Smith and Harper, 2013). The precise time point of radiation has also been under discussion (Er-win et al., 2011). Apart from environmental changes, events of major genetic transformations have been linked to the time of the Cambrian radiation, for instance the WGDs occurring in the vertebrate predecessor about 500 Mya (Nakatani et al., 2007; Putnam et al., 2008). In the vertebrate predecessor the

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genome was tetraploidized twice before the divergence of cyclostomes (lam-preys and hagfishes) and these events are referred to as 1R and 2R (Figure 3). As 1R and 2R seemed to have occurred around the time of the Cambrian ra-diation and preceded the vertebrate species divergence, it is tempting to argue that the tetraploidizations provided genetic material that enabled vertebrate specific features to evolve, such as the jaws, camera eyes, complex nervous systems etc. The WGDs provided a quadrupled pool of all of the genetic ma-terial including genes and regulatory regions. In order to explore the relation-ship between WGDs and speciation and species phenotype divergence, a brief description of WGD events and mechanisms follow below.

Figure 3. A species tree displaying the evolution of some major vertebrate and in-vertebrate chordate groups, with arrows indicating time points of the vertebrate tet-raploidizations 1R, 2R and 3R. The tree lengths are approximate, calculated by time-tree.org (Hedges et al., 2006; Kumar et al., 2017). See foot note below for comment regarding 1R and 2R in cyclostomes3.

The idea of vertebrate tetraploidizations and the role of gene duplications in evolution was first suggested by the Japanese researcher Susumu Ohno in the book Evolution by gene duplication (1970). However, his hypothesis re-mained controversial for quite some time and it was not until the development of techniques and foremost the increased availability of genome assemblies that the vertebrate tetraploidizations 1R and 2R could be confirmed, about ten years ago (Nakatani et al., 2007; Panopoulou and Poustka, 2005; Putnam et al., 2008). A classical gene family to exemplify the process of the vertebrate tetraploidizations is the Hox gene family, where one cluster is present in in-vertebrates, and four in vertebrates. The WGDs expanded the Hox family in

3 Our lab has found multiple examples of paralogons with quartets of similar chromosomal regions (Dan Larhammar and Christina A. Bergqvist, personal communication). The most par-simonious interpretation is that cyclostomes share not only 1R but also 2R with gnathostomes.

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the following manner: prior to the tetraploidizations, in the vertebrate prede-cessor, there was one cluster present. This cluster duplicated once in 1R, re-sulting in two clusters, and then once again following 2R resulting in four clusters (Holland, 2015; Larhammar et al., 2002; Sundström et al., 2008).

Following the early vertebrate 1R and 2R events occurring about 500 Mya, the predecessor of the largest and most diverse vertebrate group, the teleost fishes constituting 98% of all ray-finned fishes (Actinopterygii) underwent a third WGD, 3R about 350 Mya (Figure 3) (Jaillon et al., 2004; Nakatani and McLysaght, 2017). Similarly to the WGDs occurring during vertebrate evolu-tion, the predecessors of several groups of plants have also undergone WGDs prior to speciation and species diversification (Clark and Donoghue, 2017; Jiao et al., 2011; Murat et al., 2017). In fact, most vertebrates and flowering plants descend from an ancestor that underwent WGDs, although polyploidy is more common in plants than animals (MacKintosh and Ferrier, 2017)4.

The mechanism of WGDs and polyploidy has been suggested to be either allopolyploidy or autopolyploidy. In allopolyploidy, polyploidy arises by fu-sion of the nuclei from different species, i.e. hybridization. For instance two fertilized diploid oocytes fuse, resulting in one oocyte with two sets of chro-mosomes (Furlong and Holland, 2002). Autopolyploidy in contrast arises within the same species, i.e. the genome is spontaneously doubled, presuma-bly in the zygote, thus following meiosis there are four chromosomes present (Furlong and Holland, 2002). Overall, the most common mechanism of poly-ploidy in well characterized WGD events is allopolyploidy, although the 3R in teleost fishes is an example of autopolyploidy (Martin and Holland, 2014). Regarding 1R and 2R, the mechanism of polyploidy is not known, but auto-polyploidy has been suggested (Furlong and Holland, 2002). However, inde-pendently of the mechanism of polyploidy, it seems not to be an advantageous state for genomes to maintain, and therefore (or by natural sequence diver-gence) subsequent rediploidization occurs (Furlong and Holland, 2002; Wolfe, 2001). Because many of the WGDs occurred a long time ago, most genomes have reached a functionally diploid state again and therefore we can-not study the mechanisms of rediploidization directly. In addition, the ge-nomes have undergone changes such as translocations, mutations, gene losses

4 For discussing the evolution of genes, some useful terms are introduced. For instance, ortholog is the term used for species homologs, paralog is the term for species duplicates and ohnolog is a term used to honor Susumu Ohno, to describe paralogs originating specifically from a WGD event. In addition, a term commonly used when discussing WGDs and gene evolution is paral-ogon. A paralogon corresponds to the ancestral chromosome that was present prior to the tetra-ploidizations 1R and 2R (Coulier et al., 2000). That means that now, after 1R and 2R, there should in theory be four paralogon members, or paralogous regions, present. Hence, the paral-ogous regions are related through the WGDs and to analyze the paralogon(s) of specific gene families, i.e. to analyze the conserved synteny in a larger chromosomal region, is of importance to track the history of the gene family in relation to 1R and 2R. If the gene family of interest is found to be located within a paralogon and its members can be traced to one ancestral chromo-some, one can confirm that it has expanded through WGDs.

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and gains etc. In fact, much of the material generated during the period of WGDs has been lost and is not present in the vertebrate genomes of today (Wolfe, 2001).

The WGD events provide a reasonable account for the evolution of species diversity, this conclusion is nevertheless still debated. As already mentioned, it is indeed tempting to argue that the tetraploidization events have enabled speciation and phenotypic diversification5 such as the one observed in the early vertebrates following 1R and 2R, but as discussed by van de Peer et al. (2009) it is important to remember that the relation between the WGDs and species diversity descried in the literature is a correlation, rather than a cau-sality. A fundamental problem when studying the role of WGDs arises con-sidering extinct lineages, and how to treat them in this type of analysis (Do-noghue and Purnell, 2005). The problem is illustrated in a study by Clarke et al. (2016), where extinct species were included and no correlation between WGD and phenotypic diversity could be claimed. This problem is further ad-dressed in a recent study by Robertson et al. (2017), where the salmonid fish specific WGD event was studied in relation to species diversity. The salmonid fish ancestor underwent an additional WGD, 4R. This took place quite re-cently compared to the other vertebrate WGDs, as it is estimated to have oc-curred about 95 Mya (Robertson et al., 2017). Prior to this study it was re-ported that part of the Atlantic salmon genome is going through something referred to as delayed rediploidization, i.e. not all of its genome is rediploi-dized yet (Lien et al., 2016). Therefore, the salmonid fishes provide excellent models for studies of the rediploidization mechanism and the relation between WGD events and diversification. In 2012, Schrantz et al. proposed a model for the temporal separation between 4R and phenotypic diversity called “the WGD Radiation Lag-Time Model” (Schranz et al., 2012). This model was developed further by Robertson et al. (2017), into the “lineage-specific ohno-logs resolution” (LORe) model. Robertson et al. (2017) found that 25 % of the salmonid genome has evolved according to the LORe model. The rest has evolved according to another model, referred to as the “ancestral ohnologs resolution” (AORe). According to the AORe model, the ohnologs diverge al-ready in the salmonid ancestor, in comparison to the LORe model, where the ohnologs are diverging after the speciation event hence the AORe model gen-erates older ohnologs. Robertson et al. (2017) further found that the LORe ohnologs were specifically enriched for functions separated from the older ohnologues.

Thus, it appears that once we are able to account for time more accurately in the analyses, more precise interpretations of WGD events and phenotypic diversity might be enabled. However, despite these advancements, there are likely additional factors involved. This is illustrated by the Atlantic horseshoe

5 On this subject, I am not addressing speciation or phenotype diversification, i.e. whether the accumulation of phenotype diversification leads to speciation or not.

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crab and the American paddlefish, neither of which underwent species radia-tion following a lineage-specific WGD (Crow et al., 2012; Nossa et al., 2014). On the other hand, cichlids display the highest rates of speciation among ver-tebrates, but their predecessor did not undergo a species specific WGD (Brawand et al., 2014; Kocher, 2004).

Until now, I have addressed problems of how the environment and genetic events have shaped evolutionary history. Metaphorically, one can see the "ge-netics"6 as the inner-most part of the organism, and further wonder how this inner part allows the organism to handle the environment. In other words, what type of possible functional outcomes can the organism unfold in relation to its genetics? Finally, in evolutionary terms we could ask why the "book keeping" properties of the DNA, or the genes (Gould, 2002), have been transformed across species? The ultimate outcome of an organism’s ability to struggle with its environment is through behavior, because suitable behaviors are what de-cide whether it will find food, survive and reproduce. However, a critical prob-lem arises in that even the most basic behavior involves complex interactions, and in order to deal with the environmental interactions, multiple genes and multiple levels of genetic interaction (see footnote 5) may be required. There-fore, as stated by Robinson et al. (2008), “Genes do not specify behavior di-rectly but rather encode molecular products that build and govern the func-tioning of the brain through which behavior is expressed”. In other words, what is selected is not a specific gene, but a behavior that results from a com-plexity of genes and genetic interactions7.

With this in mind, it is reasonable to suggest that a WGD event provides a resource for behavioral changes, as innovation requires complexity and a WGD event generates genetic material for higher-level complexities to de-velop. This brings me to an important aspect of this thesis work, illustrated by a quote from Gould, (1991): “Historical origin and current utility are distinct concepts and must never be conflated.” By utility, Gould means a current adaptive function. This thesis work is an attempt to understand the evolution-ary origins of DNA sequences, or genes, but not the utility or functional roles. It is of course possible to speculate about the functional relevance in behav-ioral innovations, yet be aware that current utilities (described as functions)

6 It is important to understand that genetics signifies more than just genes. There are at least three levels of organization determining the informational mechanism of cells. The first is the classic DNA sequence (the genetic dogma), the second is the cellular milieu in which it specifies the spatial conformation of macromolecules and the third is the meta-process that shapes DNA sequences dependent on environmental effects (Thieffry and Sarkar, 1998). What this means is that although the DNA sequence is evidently fundamental, it is not causal by itself. The DNA sequence does not unidirectionally store all information required for determining a specific structure, such as the brain, or function, for instance learning and memory. 7 In these regards, the arguments by Richard Dawkins are clearly wrong, as Dawkins in his book The Selfish Gene (2006) describes genes as isolated entities driving selection and where organisms are simply bearers of the genes; claiming that genes are “selfish” in their striving for “survival”, or self-maintenance.

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could have originated from different types of adaptive functions. Gould clas-sically used the cathedral in Venice as an example of exaptation, whereas a classical example in biology confers the feathers of birds (Gould, 1991, 2002). The natural argument why birds have feathers would be to say that it enables them to fly. However, it was not for flying that feathers were initially selected. It was for body temperature regulation, and they were then exapted by birds for flying (Gould, 1991, 2002). I believe that this clarification is relevant in trying to escape the classic accounts of one gene corresponding to one func-tion.

In brief, my interest has been to investigate the evolutionary events behind genomic changes. The focus will now shift into the evolution of specific ver-tebrate gene families involved in fundamental nervous system processes, the ACh receptor gene families; the nicotinic ACh receptors (nAChRs) and the muscarinic ACh receptors (mAChRs). The ACh receptor gene families were chosen as they belong to a system classically associated with learning and memory processes. However, despite the abundance of pharmacological and genetic studies, the evolution of the nAChR and mAChR gene families has not yet been fully resolved, particularly not in relation to the vertebrate WGD events.

The nicotinic acetylcholine receptors The nAChRs are ligand-gated ion-channels (LGICs). Some of the LGIC re-ceptor types are characterized by a Cys-loop, hence forming the Cys-loop su-perfamily of receptors. The nAChRs belong to this superfamily, together with serotonin (HTR3), zinc (ZAC), GABA-A and glycine receptors. A study by Jaiteh et al. (2016) showed that the Cys-loop receptors predate the emergence of eukaryotes. However, their study also suggests that the superfamily would perhaps be more accurately referred to as the Pro-loop superfamily of recep-tors, as the characteristic Cys-loop is not conserved among all members, but instead an invariant proline in that region is (Jaiteh et al., 2016).

The nAChRs can be divided into two main groups, the nAChRs present at the neuromuscular junction (NMJ) and the nAChRs present in neurons. The NMJ receptor forms a heteropentamer and has a fixed organization of its sub-units, which are encoded by the CHRNA1, CHRNB1, CHRND, CHRNE and CHRNG genes. The subunits are assembled into a functional receptor in the following way α-δ-β-α-γ/ε, forming two binding sites for ACh in the α-δ and α-ε interfaces since the binding site is formed in the interphase between an α and non-α subunit (Figure 4A). The number of α subunits constituting the pen-tamers therefore determines the number of agonist binding sites. The γ-subunit is replaced by the ε-subunit in the shift between embryonal development and post-natal life (Missias et al., 1996). The neuronal nAChRs on the other hand can be present in many different heteropentamer constellations, or as homo-pentamers. The neuronal nAChRs include the α2-α10 and the β2-β4 subunits,

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encoded by the CHRNA2-10 and CHRNB2-4 genes, respectively. The most common nAChR homopentamer is formed by α7 subunits, whereas the most commonly expressed neuronal heteropentamer is receptors consisting of α4β2 subunits (Figure 4B). In the α7 homopentamer, five binding sites are formed for ACh, whereas the α4β2 heteropentamer can contain either two or three binding sites for ACh, depending on the subunit composition.

The nAChR protein consists of four major domains (Figure 4B). It starts with an extracellular aminoterminus domain (ECD) which contains a signal peptide and the binding domain (BD), in addition to several consensus sites for N-linked glycosylation. Then follows four transmembrane regions (TM1-TM4). Between TM3 and TM4, there is an intracellular loop domain (ICD). Finally, the protein ends with a short extracellular carboxyterminus. Except the ICD, which is quite variable between the different subunits within a spe-cies, as well as between different species, all domains remain well conserved. The characteristic Cys-loop is located in the BD, it consist of 15 highly con-served amino acid residues (Cockcroft et al., 1990) and is linked by a cysteine disulfide bond located close to the TM2-TM3 loop.

Figure 4. (A) Order of subunit assembly in the adult NMJ nAChR, and position of the ACh binding sites. Figure is reused with permission from Hurst et al. (2013). (B) Basic structure of the nAChR protein domains (left), neuronal nAChR subunit as-sembly (right) and Ach binding sites in the receptor. Figure is reused with permis-sion from Zoli et al. (2015)

The channel pore is formed by residues from the hydrophobic TM1-TM4 re-gions, with TM2 particularly lining the pore lumen to support the flow of ions whereas the TM1 and TM3-4 remain in the outer part of the pore (Bertrand et al., 1993; Miyazawa et al., 2003). Upon binding of a ligand, such as the en-dogenous ACh, the pore-lining chains start to rotate which causes interruption of the helix interactions that keep the pore closed and the pore therefore wid-ens, enabling the open conformation (Miyazawa et al., 2003; Unwin, 1995). Once ligand binding occurs, the channel opens rapidly and ions flow from the extracellular space into the cell, which causes a membrane potential shift. The mammalian nAChRs are permeable to small monovalent and divalent cations, in particular Na+ (Cohen et al., 1992; Konno et al., 1991). The channel pore

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may remain open for several milliseconds before returning to a closed state or alternatively, a desensitized state. In the high-affinity desensitized state, the receptor is inactive and no agonist can bind. Following agonist dissociation and subsequent receptor recovery, the receptor conformation returns to the low affinity resting state. The desensitization features and ligand binding of different agonists varies among the different receptor subtypes (Giniatullin et al., 2005). The HTR3 and ZAC Cys-loop superfamily members are also se-lective to cations, whereas the GABA-A and glycine receptors are selective to anions. The endogenous agonist to both NMJ and neuronal nAChRs is ACh. In theory nicotine can also bind both nAChR types, hence the name, however in practice the NMJ nAChRs have very low affinity for nicotine.

The nAChRs are expressed pre- and postsynaptically. At the NMJ, the nA-ChRs are located postsynaptically and act as fast excitatory receptors, crucial in movement processes (Lindstrom, 2003). At the presynaptic site, they serve more of a modulatory role, for instance regulating neurotransmitter release (Dani and Bertrand, 2007). In the brain the nAChR signaling is involved in processes regarding for example learning, memory, anxiety, reward, sleep and food intake. Signaling dysfunction is associated with for instance Alzheimer’s disease, depression and addiction (Court et al., 2001; Philip et al., 2010; Pidoplichko et al., 2004). As already mentioned, the most abundantly ex-pressed neuronal nAChRs are the α7 homopentamer and α4β2 heteropen-tamers (Figure 5-6) (Gotti et al., 2006; Zoli et al., 2015).

The nAChRs form a complex gene family, it has many members and the neuronal nAChR subunits can assemble into many different constellations. In addition, the expression of different subunits varies in different species, some-thing that is illustrated by for instance the α2 and α4 subunits. In rodents, the α4β2 heteropentamer is highly expressed (Figure 5). However, in primates the subunit distribution appears slightly different. The most striking difference seems to be the α2 subunit, which is scarcely expressed in rodents, whereas it in some areas in the macaque is comparable to those of the α4 subunit in ro-dents (Han et al., 2000; Ishii et al., 2005; Zoli et al., 2015) (Figure 6). The α7 and α8 subunits are commonly expressed as homopentamers but they can also form heteropentamers. The α9 and α10 subunits are expressed in the cochlea for instance, but they have not been identified in the brain (Lustig, 2006). Until recently, it was believed that the α7 subunit is only expressed as homopen-tamer nAChRs however it has now been shown to also assemble into hetero-pentamers, forming α7β2 receptors (Wu et al., 2016). Despite the presence of nAChRs in many different brain regions, some show a more restricted expres-sion pattern such as the α6β3 heteropentamer, which is expressed in the mid-brain dopamine neurons, and the α4α6β2 heteropentamer that is expressed in the striatum. In the medial habenula, the α3β4 heteropentamers are found (Fig-ure 5) (Zoli et al., 2015).

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Figure 5. nAChR distribution in the rodent brain. Figure is reused with permission from Gotti et al. (2006).

Figure 6. nAChR distribution in the human (A) and monkey (B) brains. Figure is re-used with permission from Zoli et al. (2015).

The muscarinic acetylcholine receptors The mAChRs are G protein-coupled receptors (GPCRs). They constitute the typical G-protein structure, with an extracellular N-terminal followed by seven transmembrane domains (TM1-7), separated by three intracellular (IL1-3) and three extracellular (EL1-3) loops, ending with the intracellular C-ter-minal (Figure 7). The mAChR family consists of five genes, CHRM1-5, en-coding protein M1-M5, respectively. These five family members can be sub-divided into two subfamilies, one constituting the M1, M3 and M5 receptors and one constituting the M2 and M4 receptors. The M1, M3 and M5 receptors couple to Gq/11 and the M2 and M4 couple to Gi/o. Hence, the subfamilies are characterized by different G-proteins, meaning that upon ACh binding differ-ent signal transduction cascades can be initiated followed by different out-comes. Most of the mAChR receptor subtypes are expressed in several brain regions, such as the cortex, hippocampus, striatum and substantia nigra (SN) in the rat (Bernard et al., 1992; Brann et al., 1988; Vilaró et al., 1991) with the exception of the M5 which is restricted to the VTA and the SN (Vilaró et al., 1990).

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Figure 7. The structure of the M2 (blue) and M3 (blue) receptors, with the M3 lig-and tiotropium bound. Binding sites are in orange. The mAChR receptors display the typical TM1-7 organization, which is well conserved in both subfamily types of receptors. The figure is reused with permission from Kruse et al. (2014).

The mAChR endogenous agonist is ACh, but as the receptor family name in-dicates, muscarine can also act as ligand. The side chains of the TM3-7 regions form a hydrophobic pocket, acting as orthosteric binding site for ACh. In pre-vious reports the binding pocket has been shown to contain identical amino acid residues in the M2 and M3 receptor (Haga et al., 2012; Kruse et al., 2012; Tautermann et al., 2013), the crystal structures of the human M2 and M3 re-ceptors have also been reported (Haga et al., 2012; Kruse et al., 2012). Differ-ences in the degree of amino acid conservation are however observed in the loop regions, therefore those have been targets for allosteric modulators (Christopoulos, 2002; Kruse et al., 2013, 2014).

The evolution of the ACh receptor gene families There have been previous attempts in elucidating the evolution of the nAChR gene family (Le Novère and Changeux, 1995; Le Novère et al., 2002; Li et al., 2016b; Ortells and Lunt, 1995; Tsunoyama and Gojobori, 1998), however the analyses have been hampered by the complexity of the gene family, such as different evolutionary rates among its members for instance. In addition, as the initial studies were conducted more than 20 years ago the information available was limited. Therefore, the previous studies resulted in quite differ-ent tree topologies. Also, none of the studies accounted for the evolution of the nicotinic receptors in relation to the vertebrate tetraploidizations. How-ever, in a book chapter from our lab, Lundin and Larhammar (1998) presented a proposal for the evolution of the nAChR genes in relation to 1R and 2R. During this time, evidence in favor of the vertebrate tetraploidizations started to accumulate, however still there were limitations in the data available as most of the information was collected from the data available in human in the Online Mendelian Inheritance of Man (OMIM) database.

Regarding the mAChRs, there have been no previous attempts to our knowledge to elucidate their evolution in relation to the vertebrate WGDs.

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Therefore, as the availability in data and genomes to study have increased tre-mendously in the past years, we have done an attempt to elucidate the evolu-tion of the nAChR and the mAChR gene families. In previous studies of the nAChR family phylogeny and intron positions were included in the analysis. In addition to these two approaches, we analyzed the chromosomal positions of the genes, i.e. the conserved synteny and paralogous regions in some key vertebrate species.

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Aims

The overall aim of this thesis from curiosity in neuroscience was to explore how underlying molecular mechanisms relate to behavior. Although no be-havioral research is presented in this thesis work, the molecular and bioinfor-matics work was designed with the aim to systematically characterize systems that have classically been correlated with behaviors related to reward, learning and memory.

Specific aims for each study Paper I: To investigate the methodological properties of a specific neuronal subpopulation of the VTA, defined in the TRPV1-Cre transgenic mouse line. Papers II and III: To investigate the evolution of the nicotinic and muscarinic acetylcholine receptors in vertebrates, specifically the gene family expansions in relation to the early vertebrate tetraploidizations 1R and 2R.

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Experimental procedures

Part I Animals and ethical considerations All animal experiments were approved by the Uppsala Ethical Committee (Uppsala Animal Ethics Committee, Jordbruksverket) and carried out accord-ing to Swedish regulations and European Union legislations. The transgenic DAT-Cre mouse line (Bäckman et al., 2006) was used for anatomical experi-ments and the TRPV1-Cre mouse line (Lagerström et al., 2010), crossed with the red-fluorescent Cre-reporter 129S6-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J line (Bäckman et al., 2006), was used for anatomical and functional experiments. The background of both lines was c57/bl6 129Sv. Genotyping for Cre recombinase and tdTom verification was performed as previously described (Viereckel et al., 2016).

Optogenetics – virus injection and light stimulation Adult TRPV1-Cre tdTom and DAT-Cre mice were anesthetized and stereotaxi-cally injected with a viral construct carrying the cation channel channelrho-dopsin-2 (ChR2) together with a fluorescent marker (enhanced yellow fluo-rescent protein, EYFP), or with a virus carrying the marker only (EYFP) as a control, unilaterally into the VTA. Following the injection, an optical fiber was implanted into the VTA. The mice were recovering for at least 2 to 3 weeks after the surgery, before experiments were conducted.

For functional evaluation of the ChR2-EYFP construct injection, the TRPV1-CrettdTom/ChR2-EYFP and TRPV1-CreEYFP mice received continuous light stimulation of a 473nm laser (CNI laser) for 45 minutes into the VTA, prior to sacrifice. Following perfusion with PBS and 4% paraformaldehyde (PFA), the brains were incubated in PFA over night before sucrose-treatment and freezing.

Immunohistochemical staining procedures Multi-fluorescence labelling Coronal cryo brain sections of 30 μm thickness cut in series of four (Navrati-lova et al., 2012) were prepared. The advantage of thin sections and serial

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cutting is that several separate stainings can be performed within the same area, and much information can be extracted from the material. For functional analysis of the ChR2-EYFP construct expression in the TRPV1-Cre mouse line, immunohistochemical staining against the protein encoded by the imme-diate early gene cFos was performed. The brain sections were incubated with a primary antibody, the goat anti-cFos (Santa Cruz, art nr sc-52-G). In all flu-orescent stainings performed, fluorescent staining against the EYFP expres-sion, by using a rabbit anti-GFP antibody (Abcam, art nr ab6556), or chicken anti-GFP (Abcam, art nr ab13970), was applied in order to avoid uneven photo bleach effects. The 4',6-diamidino-2-phenylindole (DAPI) was also applied in all experiments for nucleic staining. Upon secondary antibody incubation, the sections were analyzed in a Zeiss LSM520 laser scanning confocal micro-scope.

cFos co-expressing GFP neurons were analyzed, together with the expres-sion efficiency of the ChR2-EYFP in the tdTom cells, by manually counting tdTom neurons co-expressing GFP. As different viral strains, tissue types and brain regions may express and react to the opsins differently, careful charac-terization is required for correct circuit activity interpretations. It is important to analyze the temporal aspects of virus expression in the tissue if one is per-forming experiments over a longer period of time, since long-term ChR2-con-struct expression might affect the tissue (Jackman et al., 2014; Miyashita et al., 2013).

For fluorescent staining and identification of dopaminergic and glutama-tergic brain regions in whole brain sections, vibratome sections were incu-bated with guinea pig anti-VGLUT2 (Millipore art nr AB2251) and mouse anti-TH (Millipore art nr MAB318). Following secondary antibody incuba-tion, the sections were scanned in a Mirax MIDI automatic slide scanner. For fluorescent staining and identification of dopaminergic and glutamatergic syn-apses in TRPV1-Cre VTA projection areas, the 30 μm serial brain sections were incubated with mouse anti-DAT (Human Atlas Antibodies art no AMAb911125) or rabbit anti-VGLUT2 (Human Atlas Antibodies art nr HPA039226), together with chicken anti-GFP (Aves Labs cat no GFP-1020). Following secondary antibody incubation, the sections were analyzed with a Zeiss LSM520 laser scanning confocal microscope as previously described (Ippolito and Eroglu, 2010) and co-localizing synapses were manually counted and analyzed.

Immunoperoxidase staining Immunoperoxidase staining was performed on separate series of the 30 μm cryo sections. The sections were incubated with the primary antibody rabbit anti-GFP (Abcam, art nr ab6556), followed by a biotinylated anti-rabbit (Vec-tastain Elite ABC kit, Vector Laboratories) and an ABC complex before final

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treatment with a DAB Peroxidase (HRP) Substrate Kit, 3,3’-diaminobenzi-dine (Vector Laboratories). The advantage of immunoperoxidase staining over immunofluorescence staining is that it excludes the risk of photo bleach during expression pattern analysis and mapping. The sections were then scanned in a Mirax MIDI automatic slide scanner.

Part II Amino acid sequence retrieval and multiple sequence alignment Amino acid sequences were retrieved from the Ensembl or NCBI public data-bases for a group of pre-selected species. In cases where no sequence was found in either of the databases, a TBLASTN search was performed by using the sequence of a closely related species as search template. Jalview with Mus-cle default settings was used for multiple sequence alignment (Waterhouse et al., 2009). Manual editing was performed if the amino acid sequences were aligning poorly or if sequence information was lacking, whereupon the ge-nomic regions were carefully analyzed and compared to closely related spe-cies.

Phylogenetic analyses For analysis of the nAChR gene family, a maximum likelihood (ML) analysis was performed using the IQ-TREE 1.6.3 application (Nguyen et al., 2015; Trifinopoulos et al., 2016) with the ModelFinder (Kalyaanamoorthy et al., 2017) and node supports calculated by the non-parametric UltraFast Bootstrap (UFBoot) method (Hoang et al., 2018) and Shimodaira–Hasegawa approxi-mate likelihood ratio (SH-aLRT) branch test with 1000 replicates. For the mAChR gene family a ML analysis was performed using the phylogenetic maximum likelihood (PhyML) 3.0 web server (available at: http://www.atgc-montpellier.fr/phyml/) (Guindon et al., 2010) with the “Automatic Model Se-lection by SMS” option with the Akaike Information Criterion for selection of the most optimal substitution model. Note that both applications used for gen-erating ML trees used automatic selection of the substitution model most ap-propriate for each sequence alignment.

Conserved synteny and paralogon analysis of neighboring gene regions For analysis of chromosomal positions and conserved synteny of the ACh re-ceptor genes and their neighboring gene families, the corresponding genomic regions in human, chicken and spotted gar genes were analyzed. The genomic regions 10 Mb upstream and downstream of the nAChR genes in spotted gar

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was retrieved in Ensembl 83 using the Biomart function. From the resulting gene lists, gene families with at least two members present were selected for analysis of conserved synteny. Phylogenetic analysis and verification of se-quence orthology was carried out by retrieving the amino acid sequences from human, chicken, coelacanth, spotted gar and zebrafish, creating a Jalview alignment and then constructing aLRT SH-like trees with the PhyML 3.0 web server (available at: http://www.atgc-montpellier.fr/phyml/) (Guindon et al., 2010), to verify sequence orthology and identify paralogous genes. The re-gions surrounding the CHRNA2, CHRNA4 and CHRNA1 genes were already analyzed in detail by our lab (Cardoso et al., 2016; Dreborg et al., 2008; Lar-hammar et al., 2002; Sundström et al., 2008; Widmark et al., 2011) and there-fore those regions were not analyzed further in this thesis work.

For nAChR paralogon 1, the genomic regions in zebrafish, medaka, stick-leback and fugu was also investigated for analysis of gene family expansion in relation to 3R. For the mAChR gene family, the synteny analysis was per-formed in the same way as described for the nAChR genes, with the exception that the positions of the analyzed genes were also analyzed in zebrafish, in both paralogons. In addition, the gene lists retrieved based on the CHRM2/CHRM4 genes contained relatively few gene families in spotted gar, therefore the analysis was complemented with retrieval of the corresponding genomic regions based on chicken.

Intron position analysis To determine the protein domain boundaries, the Pfam (available at: http://pfam.xfam.org/) and TMHMM Server v. 2.0 (available at: http://www.cbs.dtu.dk/services/TMHMM/) web pages were used. The exon-intron organization in the nAChR genes is based on the human genes. In cases where genes are lacking in human (the CHRNB1L, CHRNB2L, CHRNA8 and CHRNA11 genes), spotted gar sequences were instead used and in the one occasion where the spotted gar sequence was incomplete (the CHRNA11 gene, lacking fours exons) zebrafish was used instead. Structurally important fea-tures (e.g. N-linked glycosylation sites and cysteines) were compared across all nAChR vertebrate orthologs included in analysis. For analyses of intron position in the mAChR teleost sequences, additional teleost species were in-cluded for genomic region comparisons.

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Results

Paper I The VTA subpopulation of neurons marked by the TRPV1-CretdTom line is mostly glutamatergic and located in the rostromedial part of the VTA, subse-quently decreasing in the caudal direction along the VTA axis, as previously reported (Viereckel et al., 2016). In this study, a virus carrying the ChR2 fused to EYFP (ChR2-EYFP) was injected into the VTA in TRPV1-Cre mice, to an-alyze the ChR2-EYFP expression extension and efferent target regions of the VTA subpopulation of neurons. The VTA subpopulation of neurons marked in TRPV1-Cre mouse is referred to as VTATRPV1-Cre in this study. Upon virus injection, the ChR2-EYFP reaches neurons in all subcompartments in the VTATRPV1-Cre population. When comparing the ChR2-EYFP expression in the TRPV1-Cre mice to the corresponding injection in a DAT-Cre mouse, it is clear that the ChR2-EYFP expression in the TRPV1-Cre mouse is restricted to a subpopulation of VTA neurons, whereas the ChR2-EYFP expression in the DAT-Cre mouse spreads throughout the VTA and the SN and is restricted to the dopaminergic neurons. Manual counting of the VTATRPV1-Cre/tdTom neurons expressing GFP, following immunohistochemical staining for virus detection, at three rostrocaudal bregma levels showed that the expression efficiency is highest in the areas in close proximity to the site of ChR2-EYFP injection into the VTA, and subsequently becomes weaker when moving away from the in-jection site. For instance, the IF displays the highest rate of expression of all VTA subcompartments, 90% of the tdTom neurons expressed GFP, as this area is located just ventral to the site on injection. The regions containing the highest numbers of TRPV1-CretdTom neurons, the supramammillary nucleus (SUM) and rostromedial ventral tegmental area (VTARM) are located in the most rostral part of the VTA, quite far from the site of injection. The overall expression efficiency in these subcompartments was approximately 50%, the same as for the entire VTATRPV1-Cre population, indicating a relatively re-stricted expression of the ChR2-EYFP in the VTATRPV1-Cre population.

Following optical stimulation, the ChR2-EYFP VTATRPV1-Cre neurons were expressing cFos in a pattern corresponding to the ChR2-EYFP expression, whereas no cFos expression was observed in mice injected with a control vi-rus, expressing EYFP but not ChR2. cFos expression was further found in the olfactory bulbs, PFC, Tu, lateral (LS) and medial septum (LS), preoptic area

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(PO), anterior hypothalamus (AH), cortical amygdala (Co), ventromedial hy-pothalamus and median raphe nucleus (MnR). Some of these regions, the ol-factory bulbs, PO and AH, contained cFos-expressing neurons also in the con-trols, therefore reflecting basal activity in these regions. However, the cFos expression in Co and MnR was specific to the ChR2-EYFP injected animals and not present in controls, suggesting that the activation of these regions re-flects a direct effect of optogenetic VTATRPV1-Cre stimulation. In the PFC, Tu and medial septum cFos expression was found in one animal per area.

Next, to identify the VTATRPV1Cre efferent target regions, mapping of the ChR2-EYFP expressing efferents was performed showing that the VTATRPV1-

Cre fibers project to the PFC, VP, BNST, CA2 layer of the hippocampus, LHb, LH, amygdalohippocampal area (AHi), MnR and the DR. Dense fibers were observed in the medial NAcSh and LS, as previously reported (Viereckel et al., 2016), and the PO. Further, one of the VTATRPV1-Cre efferent target regions was analyzed in more detail, the PFC. Immunohistochemical staining against DAT and VGLUT2 displayed a higher number of VGLUT2 positive synapses than DAT positive synapses targeting the PFC, suggesting that glutamatergic VTA efferents may have a role in modulating PFC function.

Paper II Upon collection and analysis of gene sequences from a selection of verte-brates, our phylogenetic analyses of the nAChR family suggests that there were ten ancestral nAChR genes present prior to the time point of the verte-brate tetraploidizations, which we further suggest corresponds to ten nAChR subfamilies. Eight of the ten subfamilies have either amphioxus, tunicates or both as closest relative, supporting that the nAChR expansions occurred at the time of the vertebrate tetraploidizations, 1R and 2R. The ML tree further dis-played that the CHRNA9 and CHRNA10 genes diverged first through duplica-tion, followed by the CHRNA7/CHRNA8/CHRNA11 subfamily that triplicated and display a slightly different species repertoire. The CHRNA8 gene is lack-ing in mammals, but present in chicken, lizard, frog, cartilaginous fish and ray-finned fish. In addition, a previously undescribed gene was identified, CHRNA11 which is present in lizard, coelacanth, cartilaginous fish and ray-finned fish.

Next, the ancestor to the NMJ nAChR subfamilies CHRNB1/CHRNB1L, CHRND, CHRNE/CHRNG and the CHRNB2/CHRNB2L/CHRNB4 subfamily branched off. CHRNB1 and CHRND are single genes, however instead a local duplicate of CHRNB1, the CHRNB1L (L for like) gene is present in ray-finned fishes. The CHRNB1 gene is lacking in chicken, lizard, frog and Australian ghostshark but found in additional reptile genomes such as turtle, python and alligator. The CHRNE gene is also lacking in chicken, but just as CHRNB1, it is found in additional reptile genomes. The ancestor to the CHRNB2/

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CHRNB2L/CHRNB4 subfamily triplicated. However, the CHRNB2 gene is lacking in spotted gar, which instead has the CHRNB2L gene. The CHRNB2L gene shows a different phylogenetic distribution overall, as it is present in Australian ghostshark and ray-finned fish only.

Next in the ML tree follows the divergence of the rest of the α-subunit genes, where the first to branch off is the CHRNA1 gene, that has remained single. Despite the function as a NMJ-subunit, the CHRNA1 gene clusters to-gether with the rest of the neuronal α-subunit genes. Notably, CHRNA1 has a local duplicate present in frog. The rest of the neuronal α-subunit genes dupli-cated, forming three separate pair subfamilies namely the CHRNB3/CHRNA5, CHRNA3/CHRNA6 and CHRNA2/CHRNA4.

In summary the phylogenetic analysis showed that the ten ancestral nAChR genes expanded into 19 genes, of which three were lost in mammals (the CHRNA8, CHRNA11 and CHRNB2L genes) resulting in 16 genes present to-day. The vertebrate species repertoire as well as the sequence presence in in-vertebrate chordates argues strongly that the expansion of ten nAChR genes into 19 occurred at a time in consistence with the 1R and 2R. In addition, the nAChR repertoire in the teleost fishes increased from 20 genes (including the CHRNB1L local duplicate) into 31 genes present in the teleost ancestor, of which 27 nAChR genes are present in zebrafish (with paralogs of the CHRNA2, CHRNA4, CHRNA7, CHRNA9, CHRNA10, CHRNB2L, CHRNB3 genes). The phylogenetic analysis suggests that the timing of the teleost nA-ChR gene duplications took place at the time of the teleost specific tetraploidi-zation, 3R. Therefore, it seems likely that the teleosts increased their repertoire also through a tetraploidization event. In this study, zebrafish, medaka, stick-leback and fugu were included as teleost representatives. Interestingly, the only genes found to have retained the 3R duplicates in all species were the CHRNB3, CHRNA9 and CHRNA10 genes, whereas the CHRNA3, CHRNA5, CHRNB2, CHRNB4, CHRNB1, CHRNB1L, CHRND, CHRNE and CHRNG genes retained no 3R duplicates in any of the species investigated. The rest of the nAChR genes have retained a 3R duplicate in at least one of the species investigate. The phylogenetic results of subfamily division are also supported by analysis of the exon-intron organization in the nAChR genes. Notably, in depth analysis of the NMJ genes verified that the CHRNA1 gene appears to be more closely related to the neuronal α-subunit genes rather than the NMJ genes also based on intron positions, as no evidence strong enough to indicate otherwise was found.

In order to verify the nAChR gene family expansion through 1R and 2R suggested by the phylogenetic analysis, in depth chromosomal synteny and paralogon analysis of the nAChR genes and their neighboring genes was per-formed. This analysis confirmed expansion of the nAChR family following the vertebrate tetraploidizations. The analysis shows that the nAChR genes belong to five different paralogons. Two of the paralogous regions have been studied in detail by our lab previously (Cardoso et al., 2016; Dreborg et al.,

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2008; Larhammar et al., 2002; Sundström et al., 2008; Widmark et al., 2011). None of the neighboring gene families showed a pattern of evolution that would disagree with expansion through 1R and 2R. A similar analysis in the teleost fishes verified that the nAChR family most likely expanded thorough 3R in teleost fishes also. In summary, our analyses which are based on a com-bined approach including phylogeny, intron positions and conserved synteny, show that the nAChR gene family consists of 10 subfamilies, corresponding to the ten ancestral genes present prior to 1R and subsequently increased to 19 genes following the 1R and 2R events. Three additional genes were lost in mammals, resulting in 16 subunit genes present in humans today, 15 in chicken and 19 in spotted gar (including the local duplication of the CHRNB1 gene). Following the teleost tetraploidizations, 20 genes present in the teleost predecessor expanded to 31 nAChR genes present in the teleost ancestor. Sub-sequently, 27 nAChR genes are present in zebrafish today, 28 in medaka, 27 in stickleback and 28 in fugu. Therefore, all vertebrate duplication events in the nAChR gene family except two (one in the ancestor of ray-finned fish and one in the frog Xenopus tropicalis) occurred through WGD events.

Paper III The sequence-based PhyML analysis of the mAChR gene family showed that two ancestral genes were present in the vertebrate predecessor, each of which giving rise to one of the two mAChR subfamilies following duplication events. One ancestor gene duplicated forming the CHRM2 and CHRMN4 genes, and the other triplicated forming the CHRM1, CHRM3 and CHRM5 genes. All mAChR gene family members were found in all species investi-gated except the CHRM1 gene, which could not be identified in the chicken genome, or any other bird, nor in medaka or stickleback. Interestingly, all tel-eosts included in the analysis (European eel, zebrafish, medaka and stickle-back) have retained duplicates of all mAChR genes, except European eel where one copy only was identified for the CHRM2 gene, and as already men-tioned the CHRM1 genes that are lacking in medaka and stickleback.

Overall, the mAChR sequence alignment revealed a high degree of se-quence identity. The TM regions displayed the highest degrees of sequence conservation, where a pairwise alignment of human and one of the most slowly evolving vertebrate species, spotted gar, displayed 96% sequence iden-tity for the CHRM2 gene, which is the gene that had retained the highest de-gree of conservation. The gene displaying the lowest degree of sequence con-servation is the CHRM1 gene, with 83% sequence identity. However, if com-paring the complete sequences in a pairwise alignment, the sequence identity for CHRM2 drops to 75%. This is due to the low degree of conservation in the intra- and extracellular loops. In particular, a part of the IL3 contains a region of low sequence conservation, both when comparing separate genes as well as

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different species. Therefore, the variable part of the IL3 region was removed in the sequence alignment for the PhyML analysis to be carried out. This in-creased the sequence identity of CHRM2 to 87%, although it does not reach the percentage as the one observed when looking at TM regions only, it in-crease substantially which clearly displays the low degree of sequence con-servation in the IL3 region. However, despite the exclusion of a highly varia-ble part of the alignment, some of the bootstrap support values in the PhyML tree remain low. An example is the nodes at the divergence of tunicate se-quences. The position of tunicates in the tree does not appear stable, some of them have also evolved fast which is indicated by their long branches. There-fore, the fact that the CHRM2/CHRM4 subfamily lacks tunicates as closest relative, might not mirror the actual phylogeny. Additional sequences showed varying evolutionary rates, despite the IL3 region exclusion. This was ob-served especially in the teleost fishes. Upon closer inspection of these se-quences, it was found that some of the teleost genes contain introns, despite previous reports on mAChR genes to be intron less (Bonner et al., 1987, 1988; Peralta et al., 1987; Seo et al., 2009). In depth analyses with support from additional teleost species showed that the CHRM2b, CHRM3b, CHRM4a and CHRM4b genes have independently gained for instance one intron at least in the TM1 region and at least one intron in the IL3 region. Some genes also gained introns in the TM3 and TM5 regions. The CHRM4a gene has gained the highest number of introns, six in total; one prior to TM1, one around TM5 and no less than four introns in the IL3 region.

In order to investigate the hypothesis resulting from the phylogenetic anal-ysis, whether the mAChR family had expanded through the vertebrate tetra-ploidizations or not, conserved synteny and paralogon analysis of the mAChR genes and their neighboring genes was performed. Analysis of the genomic regions surrounding the mAChR genes showed that the mAChR genes belong to two separate paralogons, one per subfamily and the analysis supported the phylogenetic results of gene family expansion from two ancestral genes to ten mAChR genes following 1R and 2R. Some genomic blocks had been translo-cated to other chromosomes, as commonly seen following tetraploidization events. In particular, the genomic regions investigated in zebrafish had been subject to several translocations, however these chromosomal regions have been carefully investigated by our lab previously and shown to originate from 3R (Lagman et al., 2013; Ocampo Daza et al., 2012). Finally, the synteny analysis confirms the hypothesis resulting from the phylogenetic analyses; the two ancestral mAChR genes duplicated in 1R and 2R, resulting in five mAChR genes. In teleosts fishes, 3R resulted in all paralogs retained in zebrafish, and a total of ten mAChR genes.

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Discussion

The VTATRPV1-Cre population as a model for investigating VTA function (Paper I) The VTATRPV1-Cre population is restricted to a subpopulation of the VTA, with-out spreading to neighboring regions (Viereckel et al., 2016). Therefore, it provides a unique opportunity for disentangling the VTA complexity. Alt-hough the neuronal types of the subpopulation has been characterized previ-ously (Viereckel et al., 2016), the connectivity and VTATRPV1-Cre efferent target regions has not been fully analyzed. Mapping of the VTATRPV1-Cre efferent tar-gets following injection of ChR2-EYFP reveals some interesting regions in comparison to what is enabled by the corresponding injection in the DAT-Cre mouse. As neurotransmitter based Cre-lines such as the DAT-Cre, VGLUT2-Cre and GAD-Cre have commonly been used in studies of the VTA, the TRPV1-Cre mouse offers region specific studies, and more specifically region specific studies of the non-dopaminergic population of the VTA. As studies of the non-dopaminergic VTA compartments have traditionally used the VGLUT2-Cre and GAD-Cre mouse lines, where the Cre expression extends the VTA borders, the TRPV1-Cre mouse line offers an opportunity to specifi-cally study the non-dopamine VTA population, overcoming technical chal-lenges that a wide Cre-expression might cause for instance in the application of optogenetics.

The analysis further shows that when evaluating the expression efficiency of ChR2-EYFP in the VTATRPV1-Cre/tdTom neurons, 50% of the population ex-presses ChR2-EYFP. There are two possible explanations for this relatively low rate of expression. First, the expression efficiency displays that the highest rates of expression are found in VTA subcompartments in close proximity to the site on injections, i.e. it is possible that the ChR2-EYFP does not reach VTATRPV1-Cre/tdTom neurons located far from the injection site as efficiently as the ones in close proximity. Second, TRPV1 expression is detected mainly during development as the adult mice show low expression levels (Viereckel et al., 2016), which could mean that due to a switch in TRPV1 expression dur-ing development, the neurons are still expressing tdTom in the adult mouse, but not the Cre-recombinase and therefore they do not express ChR2-EYFP upon injection of the virus construct. Although this analysis is based on one animal only and should be repeated in a higher number of animals for general

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conclusions to be drawn, it highlights the importance of analyzing the expres-sion efficiency in virus- and transgenic mouse based approaches. In addition, it demonstrates the importance of careful characterization of the specific cir-cuit investigated when using a transgenic line such as the TRPV1-Cre contain-ing a mix of neuronal classes, to know what neurons that are finally expressing the ChR2-EYFP, for interpretation of the circuit activity and downstream ef-fects.

Further, mapping of the VTATRPV1-Cre efferents showed that regions tar-geted includes the NAcSh and LS, as previously described (Viereckel et al., 2016), and the BNST, LHb and PFC, among others. These three latter ones would be of particular interest for future studies. The BNST and its relation to the NAcSh, as discussed in the introduction in this thesis, has been debated, and both structures are targeted by the VTATRPV1-Cre population. The BNST has, despite its slightly controversial anatomy, been shown to regulate stress and social behaviors (Lebow and Chen, 2016). The LHb on the other hand seems to be positioned in a way that it models midbrain monoamine activity, and has been linked to neuropsychiatric disorders, for instance depression (Lecca et al., 2014). Finally, the PFC is perhaps the most interesting candidate among all since the PFC has been shown to be co-activated with the VTA in the theta-frequency during a working memory task associated with reward (Fujisawa and Buzsáki, 2011). The authors of the study suggest that a mecha-nism for this could be the fast glutamatergic signaling. Indeed, in my study cFos expression was found in the PFC following optogenetic stimulation of the VTATRPV1-Cre neurons. Furthermore, fluorescent stainings showed that a higher proportion of the VTATRPV1-Cre PFC synapses were VGLUT2 positive rather than DAT. Therefore, despite the sparse fibers these results suggests the predominance of an excitatory output from the VTA to the PFC. Finally, again referring back to the introduction of this thesis, the VTA-PFC subcircuit iden-tified in this study provides an example of brain areas involved in both emo-tional and cognitive processes. Overall, the TRPV1-Cre mouse line offers a highly useful tool for studying the VTA complexity, combined with the use of optogenetic tools.

The evolution of the nAChR gene family and its expansion in the vertebrate WGDs (Paper II) To undertake the complexity of the nAChR family and its evolution, and fac-tors that have previously hampered analysis, we used a combined approach including sequence-based phylogeny, exon-intron organization and conserved synteny and paralogon analyses in order to elucidate the gene family evolu-tion. This combined approach of analyses suggests that there were ten ances-tral nAChR genes present prior to the tetraploidizations, which expanded into

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19 following 1R and 2R. Three of these were lost in mammals (the CHRNA8, CHRNA11 and CHRNB2L genes). Only two local duplications were identi-fied, one of the CHRNB1 gene in the ray-finned fish ancestor, forming CHRNB1L, and one duplication of CHRNA1 in the frog Xenopus tropicalis. Apart from these two events, all other vertebrate duplication events were re-lated to 1R and 2R.

The ten nAChR ancestral genes correspond to ten nAChR subfamilies. Two of the subfamilies have retained three ohnologues each, namely the CHRNA7/CHRNA8/CHRNA11 and the CHRNB2/CHRNB4/CHRNB2L clades. Most of the nAChR ancestral genes retained two ohnologs following 1R and 2R, whereas three retained only one and therefore remained single genes. Hence, none of the ancestral genes kept all four ohnologs following 2R, but losses are common following WGD events. In fact, neither can we here differentiate between the 1R and 2R in some circumstances, i.e. we can-not say which genes that derive from which tetraploidization event. For genes that have kept all four ohnologs, it means that the ancestor gene duplicated following 1R and then both resulting copies duplicated again following 2R, resulting in four ohnologues. However, for pairs we cannot know whether one of the duplicates following 1R was lost and the remaining copy was duplicated in 2R resulting in the two ohnologues present, or whether no copies were lost following 1R but instead both 2R copies were lost. Neither can we know from which tetraploidization event that single genes remain.

In previous studies on the evolution of the nAChR gene family, the evolu-tion of some of the genes was particularly difficult to resolve. For instance, the relationship of the CHRNA1 gene to the other nAChR genes has shifted. Initially, it was suggested to cluster together with the neuronal subunit genes (Le Novère and Changeux, 1995), but in a later study it was instead clustering with the rest of the NMJ genes (Le Novère et al., 2002). Our phylogenetic analyses, including a broad range of vertebrates, shows that the CHRNA1 gene clusters together with the neuronal α-subunit genes. However, we found that when analyzing the exon-intron organization of the genes CHRNA1 shows similarities to the other NMJ genes, but upon closer inspection we could not find that they are similar enough to argue against the phylogenetic results, and we therefore conclude that CHRNA1 is more closely related to the neuronal α-subunit genes than the NMJ genes. Further looking into the NMJ genes, we suggest that CHRND diverged before the appearance of the CHRNE/CHRNG genes, as a local duplication prior to 1R. Then, following 1R and 2R, no addi-tional CHRND ohnologs were retained. This is supported by an averagely low rate of retention of ohnologs among the NMJ genes. Also, the CHRNB1 and CHRNE genes have not been identified in the chicken, just as many of their neighboring genes. Although these genes could be missing in chicken, it seems unlikely, as these genes are required to form the NMJ heteropentamer. In addition, these genes are present in reptiles hence increasing the likelihood of presence also in chicken. Bird genomes contain microchromosomes, which

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are GC-rich hence difficult to sequence (Burt, 2002; Zhang et al., 2014). It is therefore possible that the genes that have not yet been identified in the chicken genome are located on microchromosomes. Further, a brief analysis of the teleost nAChR paralogs verified expansion through 3R. Hence, the 20 nAChR genes (including the CHRNB1L local duplication) present prior to the teleost specific tetraploidization increased to 31 in the teleost ancestor, of which 27 are retained in zebrafish.

All vertebrate mAChR genes originate from the vertebrate WGDs (Paper III) In this study, a similar approach was used as in the study of the nAChRs, ex-cept that the analysis was initially focused on sequence-based phylogeny and conserved synteny, since the mAChRs have commonly been said to be intron-less. However, intron analysis was later included as it was found that some of the teleost genes had gained introns. Our combined approach shows that the vertebrate mAChRs present today expanded entirely through the vertebrate tetraploidizations, no single local duplication event was identified in the mAChR gene family. In addition, all mAChR members were identified in all species investigated, with a few exceptions only regarding the CHRM1 gene. The CHRM1 gene has not been found in chicken, or any other birds. When analyzing the chromosomal regions it turned out that the entire paralogon member is lacking in chicken. However, as described in the discussion on Pa-per II, it could be that these genes are located on microchromosomes and therefore have not been identified yet. Interestingly, one chicken gene in the paralogon member was identified, but found to be located on a scaffold. In addition, medaka and stickleback lacks the CHRM1 gene, but two CHRM1 gene paralogs are present in zebrafish. Hence, despite a few exceptions re-garding CHRM1, the CHRM genes display a relatively stable species reper-toire. In fact, zebrafish has retained paralogs of all mAChR genes, resulting in ten mAChR genes present, in line with has been reported previously (Seo et al., 2009).

As briefly mentioned, our analyses identified a number of intron gains in the coding regions of the CHRM2b, CHRM3b, CHRM4a and CHRM4b genes in some teleost fishes. Due to the increased availability and quality in the tel-eost genome availability, we were able to determine the position of most of these introns. This finding is also supported by previous reports regarding in-tron gains and their relative high rate in actinopterygii, especially in stickle-back and zebrafish (Ravi and Venkatesh, 2018; Venkatesh et al., 2014). In summary, our analysis found that there were two ancestral mAChR genes pre-sent in the vertebrate predecessor. These genes increased to in total five mAChR genes following 1R and 2R, divided into two subfamilies whereas

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zebrafish retained a doubled mAChR repertoire following 3R, resulting in ten mAChR genes.

The evolution of receptor gene families (Paper II and III) To conclude whether genes have arisen through a local gene duplication or a WGD event is crucial as it makes a difference in what was duplicated with the gene, i.e. in a WGD event it is not only the gene itself or a smaller segment as in a local duplication that is duplicated, but the entire chromosome on which the gene is located. This means that along with the gene, regulatory elements including promoters, enhancers etc. are duplicated which might enable differ-ent evolutionary outcomes. Because with time, the ohnologs resulting from the WGD might accumulate genetic changes, such as mutations. This sup-poses two possible scenarios. First, one of the ohnologs might maintain the initial function of the gene, which enables the other ohnolog to evolve into other functional outcomes, also called neo-functionalization. Second, the two ohnologs might divide the initial function between them by for instance al-tered influence from regulatory elements, leading to what is called sub-func-tionalization. However, from the studies conducted during this thesis work, there can be no inferences about the function, and neo- or subfunctionaliza-tion, of neither the nAChR nor the mAChR genes. The studies conducted dur-ing this work have focused entirely on elucidating the evolution of the two gene families, which as just mentioned is of importance e.g. for understanding the regulatory mechanisms of the genes, or to interpret the results from mouse knock out (KO) studies. For instance, it is often the case that KO mice of sin-gle genes do not demonstrate an altered phenotype. This could be due to the presence of another gene that is evolutionarily closely related to the gene that is knocked out, and which could compensate for the gene loss in order to main-tain the functional outcome. In fact such an experiment, investigating possible sub- or neo-functionalization of the nAChR and mAChR genes, would be the natural experimental continuation of this evolutionary analysis. Ultimately, this could explain why an organism has acquired specific molecular changes in terms of natural selection (Mayr, 1961) and could further help us to under-stand ultimate causes.

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Conclusions

The work conducted in this thesis work covers two broad topics. First, in the study of the brain reward system, application of optogenetic methods to the TRPV1-Cre transgenic mouse line was validated as a model to investigate VTA function. A number of interesting VTATRPV1-Cre efferent target areas were identified, such as the BNST, LHb and PFC. The functionality of this trans-genic line for optogenetic studies was demonstrated as optogenetic stimulation of the VTA led to cFos expression in the ChR2-EYFP expressing VTATRPV1-

Cre neurons, as well as in some of its efferent target areas. A possible excitatory VTA to PFC subcircuit was identified in the TRPV1-Cre mouse, as cFos cells positive cells were found upon VTA optogenetic stimulation, and a higher proportion of VGLUT2 rather than DAT VTATRPV1-Cre efferent fibers were identified in the PFC. The model evaluated in this study could also be applied for in depth studies in other VTA subcircuits, such as the VTA to LHb or BNST, making the TRPV1-Cre transgenic line a highly useful tool for studies of mainly non-dopaminergic VTA subpopulation efferents.

Second, the evolution of the nAChR and mAChR gene families was dis-sected, in relation to the early vertebrate WGDs. In comparison to previous attempts of elucidating the evolution of the nAChRs, a combined approach was used to investigate this complex family. By using this approach, we could verify our hypothesis from three different sets of data. The analyses of the nAChR family concluded that ten nAChR genes were present prior to 1R, and these expanded into 19 genes following 1R and 2R, of which 16 are present in human. All duplication events could be confined to the time of the 1R and 2R, except one local duplication. The same type of approach was applied to the mAChR gene family, which is a smaller family with fewer members, how-ever still it had not been elucidated how the gene family expanded in relation to the vertebrate tetraploidizations. Our combined analysis approach con-cluded that the mAChR family expanded from two ancestral genes into five in 1R and 2R. Further, 3R resulted in paralogs of all mAChR genes in the zebrafish. Hence, all duplication events in the expansion of the mAChR family were confined to 1R, 2R and 3R.

Finally, although this thesis work took a different turn from what was ini-tially planned, the broad approaches offered by the work have helped the de-velopment of each part. The second part enabled me to explore evolutionary problems and thereby work from a different perspective than what I was used to, however during this work the achievements from the first part of the thesis

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work were of importance when trying to understand the theoretical problems of brain evolution and genetic change, in relation to the final outcome; behav-ior.

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Future perspectives

The TRPV1-Cre transgenic line offers many possibilities for future experi-ments and dissection of the reward system in mice. As already mentioned, an interesting candidate is the VTA to PFC subcircuit that the TRPV1-Cre line enables studies of. First, the anatomical characterization could be extended by further dissection of the circuitry, e.g. including characterization of which class of PFC neurons that is targeted by the VTATRPV1-Cre efferents. The next step to follow would be functional studies to investigate how certain forms of activity might relate with behavior, using optogenetic tools. An important step prior to behavioral experiments would be to characterize the PFC neurons tar-geted by the TRPV1-Cre VTA efferents electrophysiologically, to determine postsynaptic responses. It would also be of interest to investigate the subcir-cuitries between the VTA and LH, BNST, cortical amygdala, NAcSh etc. Fi-nally, a rabies-virus based approach could be applied to analyze the afferents to the VTATRPV1-Cre population to see what those results would show in relation to previous studies on VTA afferents (Beier et al., 2015; Faget et al., 2016; Watabe-Uchida et al., 2012), as different connectivity patterns have great im-pact on the behavioral outcome (Figure 2).

Regarding the evolutionary studies of the nAChR and mAChR gene fami-lies, the next step would be to move from purely bioinformatics methods into functional experiments in order to investigate possible neo- or sub-function-alization of genes arising from 3R in zebrafish. As teleosts constitute one of the most diverse vertebrate groups, they serve as good candidates for studying the relationship between a WGD event and functional implications. Zebrafish is also one of today’s most common model organisms in scientific labs, and the acetylcholine receptors are broadly expressed and involved in many pro-cesses. Hence a better understanding of the how and why of these genes will be useful for our knowledge of physiology and behavior. As presented in the introduction, differences in expression of the nAChR subunit genes are ob-served in for instance rodents and primates (Figure 5-6) (Gotti et al., 2006; Han et al., 2000; Ishii et al., 2005; Zoli et al., 2015), therefore it would be interesting to know more about the nAChR expression also in the zebrafish brain. Previous studies have investigated some of the nAChR genes in zebrafish (Ackerman et al., 2009; Menelaou et al., 2014; Zirger et al., 2003), however at the time of these studies information regarding some of the zebrafish paralogs was still incomplete, hence it would be interesting to look

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into the zebrafish nAChR paralogs based on the evolutionary analysis con-ducted during this thesis work.

A first step would be to verify gene expression levels and patterns by qPCR and in situ hybridization, then move on to behavioral experiments and make use of the e.g. the CRISPR-Cas system (Li et al., 2016a), to see how the mo-lecular properties of these genes may relate with behavior and function. An elegant example of this type of study is provided by Moriyama et al. (2016), where sub- and neo-functionalization of the zebrafish elastin paralogs in the fish heart are analyzed8. Equivalent experiments could be carried out with the ACh receptor genes in zebrafish, in order to evaluate their functional role and how differences and similarities in sequences and genome locations may re-late with specific sub- and neo-functionalization.

8 Moriyama et al. (2016) showed that the elastin b (elnb) gene, a teleost specific extracellular matrix gene, originates from 3R and through neo-functionalization contributed to the origin of the bulbus arteriosus (BA), an evolutionary novel organ advancing the teleost heart. The order of work was first bioinformatics analyses to verify the evolution of the elastin genes, followed by anatomical localization and timing of cell differentiation to find that the elnb gene neo-functionalized and lastly to perform a knockdown experiment, which showed that knockdown of elnb cause severe hypoplasia of the BA. Hence, this study elegantly linked a gene originating from a WGD event, to phenotypic diversity.

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Acknowledgement

I would like to acknowledge the people enabling the work included in this thesis to be carried out. First, I would like to thank my supervisor Professor Dan Larhammar, for taking me on as a student at a time when my future in research was not as clear as when I started, and for introducing me to research based on evolutionary questions. I would also like to thank my co-supervisor Professor Bryndis Birnir for support and for always inviting to a discussion.

Regarding the work in Paper I, I would like to most sincerely thank Dr Jan Mulder, for giving input, sharing ideas and material to enable completion of the work at a time when there were no resources available. Regarding the work in Paper II and III, I would like to thank my colleague and co-author Christina Bergqvist. Without your methodological and intellectual contribution, I am not sure I would have made it through the bioinformatics metamorphosis.

I would like to thank all people that have been involved in or enabled the work in this thesis; collaborators and past and present colleagues.

Finally, thank you to my friends, och till min familj, för att ni är de ni är.

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Acta Universitatis UpsaliensisDigital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1472

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A doctoral dissertation from the Faculty of Medicine, UppsalaUniversity, is usually a summary of a number of papers. A fewcopies of the complete dissertation are kept at major Swedishresearch libraries, while the summary alone is distributedinternationally through the series Digital ComprehensiveSummaries of Uppsala Dissertations from the Faculty ofMedicine. (Prior to January, 2005, the series was publishedunder the title “Comprehensive Summaries of UppsalaDissertations from the Faculty of Medicine”.)

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