Comparing the serotonergic system in vertebrates and invertebrates

Linköping University | Department of Physics, Chemistry and Biology

Bachelor thesis, 16 hp | Chemical Biology programme: Physics, Chemistry and Biology Spring term 2017 | LITH-IFM-X-EX--17/3339--SE

Comparing the serotonergic

system in vertebrates and

invertebrates

Elin Hessling

Examinator, Jordi Altimiras, IFM Biologi, Linköpings universitet Tutor, Hanne Løvlie, IFM Biologi, Linköpings universitet

1 _{Denna rapport är ett examensarbete på kandidatnivå (16 hp) där den experimentella delen har genomförts i}

samarbete med två studentkollegor, Simon Björklund Aksoy och Kristoffer Lundgren. Samarbetet har omfattat projektetplanering samt insamling och bearbetning av data, medan studenterna var för sig har författat och strukturerat rapporten i alla dess delar.

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Department of Physics, Chemistry and Biology Linköping University

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ISRN: LITH-IFM-G-EX--17/3339--SE

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Språk Language Svenska/Swedish Engelska/English ________________ Rapporttyp1 Report category Licentiatavhandling Examensarbete C-uppsats D-uppsats Övrig rapport _____________ Title

Comparing the serotonergic system in vertebrates and invertebrates

Author

Elin Hessling

Nyckelord

Serotonergic system, serotonin receptors, SSRIs, serotonin transporter protein Abstract

The serotonergic system is involved in a broad range of functions in both vertebrates and

invertebrates and is highly conserved across taxa. Serotonin is an important monoamine acting in the brains of humans and animals, and has large and varying influences on many aspects of an individual’s life. For example, in humans, serotonin modulates feelings of happiness and in fruit flies, higher levels of serotonin increase aggression. In humans, an abnormal serotonergic system can result in health issues, such as depression and obsessive compulsive disorders, for which medications have been developed, including selective serotonin reuptake inhibitors (SSRI). Because the serotonin system has a large influence on human health, understanding how it

functions is of great interest to researchers. Using comparative studies to explore differences in the serotonin system across taxa can provide insight into the mechanistic details of the system. To investigate if the serotonin system is comparable between vertebrates and invertebrates, a literature study with particular focus on receptors and proteins involved was performed. In addition, this report takes part in an experimental study investigating the effect of the SSRI fluoxetine in Mediterranean field crickets. Fluoxetine reduced exploration propensity of crickets, which was reversed, compared to what was anticipated and compared to effects seen in vertebrates. The literature review suggests that serotonin receptors are quite similar, but that proteins differ more when comparing vertebrates and invertebrates. This offers a likely explanation as to why results of studies on these different groups of animals may differ.

Table of contents

1. Abstract ... 1

2. Introduction ... 1

3. Literature review ... 4

3.2.1 Biosynthesis and metabolism of serotonin ... 4

3.2.2 Receptors involved in the serotonergic system ... 4

3.2.3 Serotonin receptor subtypes in vertebrates... 5

3.2.4 Serotonin receptor subtypes in invertebrates ... 5

3.2.5 Serotonin transporter proteins ... 6

3.2.6 Serotonin transporter protein in vertebrates and invertebrates ... 7

3.2.7 Blocking the serotoninergic system ... 8

3.2.8 Fluoxetine ... 8

4. Experiment ... 9

4.1 Material and Method ... 9

4.1.1 Subjects ... 9

4.1.2 Treatment ... 9

4.1.3 Exploration ... 9

4.1.4 Statistics ... 10

5. General Discussion ... 11

5.1 Social and ethical aspects ... 11

5.2 Conclusion ... 12

Acknowledgement ... 12

References ... 13

1

1. Abstract

The serotonergic system is involved in a broad range of functions in both

vertebrates and invertebrates and is highly conserved across taxa. Serotonin is an important monoamine acting in the brains of humans and other animals, and has large and varying influences on many aspects of an individual’s life. For

example, in humans, serotonin modulates feelings of happiness, and in fruit flies, higher levels of serotonin increase aggression. In humans, an abnormal serotonergic system can result in health issues, such as depression and obsessive compulsive disorders, for which medications have been developed, including selective serotonin reuptake inhibitors (SSRI). Because the serotonin system has a large influence on human health, understanding how it functions is of great interest to researchers. Using comparative studies to explore differences in the serotonin system across taxa can provide insight into the mechanistic details of the system. To investigate if the serotonin system is comparable between

vertebrates and invertebrates, a literature study with particular focus on involved proteins such as receptors and transporters, was performed. In addition, this report contains an experimental study investigating the effect of the SSRI fluoxetine in Mediterranean field crickets. Fluoxetine reduced exploration propensity of crickets, which was reversed compared to what was anticipated and compared to effects seen in vertebrates. The literature review suggests that serotonin receptors are quite similar, but that the serotonin transporter protein differ more when comparing vertebrates and invertebrates. This offers a likely explanation as to why results of studies on these different groups of animals may differ.

2. Introduction

The monoaminergic system is a complex system regulating many aspects of functions in the body. Monoamines such as dopamine, histamine and serotonin regulate different behaviours, for example, aggression, sleep, activity and learning (Blenau & Baumann 2001). One of the well-studied monoamines is serotonin (Figure 1), which is found in all organisms that have a central nervous system. In humans, serotonin affects feelings of well-being, self-confidence and happiness. Fruit flies (Drosophila) with increased levels of serotonin show higher fighting frequencies and more intense fighting than ones with lower levels (Dierick & Greenspan 2007). Evolution of the serotonergic system is highly conserved and exists in a broad range of species, including both

vertebrates and invertebrates. This suggests that the serotonergic system may have an important role in allowing organisms to alter their behaviour traits (Lövheim 2012). Humans and fruit flies seem to have similarly functioning

2 serotonergic systems. The first functions shown to be similar were sleep and appetite (Yuan et al. 2006). The serotonergic system is dependent on pre- and postsynaptic neurons, receptors and transporter proteins. Receptors and

transporters involved in the system are two of the major keys to making the serotonergic system work (Strüder & Weicker 2001).

Serotonin originates from the essential amino acid tryptophan which is commonly found in most proteins. Serotonin belongs to the group of natural products that have a basic amine group separated from an aromatic core by an aliphatic chain consisting of two carbon oxides (Figure 1).

Figure 1. Chemical structure of serotonin.

The metabolic precursor of serotonin (5-Hydroxytryptophan, 5-HTP) is involved in the biosynthesis of serotonin in vertebrates. 5-HTP is also part of the

biosynthesis of the serotonergic system in invertebrates (Dyakonova et al. 2012). The pathway of serotonin biosynthesis for both vertebrates and invertebrates requires several steps (see Figure 2). These steps involve addition of a hydroxyl group to the indole ring and a decarboxylation. An enzyme, tryptophan

hydroxylase (TPH), is necessary for the process. The role of TPH is to add a hydroxyl group to tryptophan. TPH uses O2 and tetrahydrobiopterine as its

cofactors to catalyse the reaction. In the second step of the synthesis, the 5-HTP is decarboxylized by an enzyme called HTP decarboxylase which converts 5-HTP to 5-HT (Vleugels et al. 2015).

3 Figure 2. Overview of the biosynthesis of serotonin in both vertebrates and

invertebrates (Vleugels et al. 2015).

To be able to act in the body, serotonin has to be stored. Biogenic amines, such as serotonin, are stored in the synaptic terminals to be released at chemical synapses. Serotonin binds to specific receptor proteins. There are at least 14 serotonin receptors that are coupled into seven different groups according to their signalling mechanism, structure and function in humans (Kroeze et al. 2002). Another important aspect of the serotonergic system in vertebrates is the serotonin transport protein, SERT. SERT is a transporter that is located on the membrane of the presynaptic cell and regulates the reuptake of serotonin from the synaptic cleft (UniProt 2017). Selective serotonin reuptake inhibitors (SSRIs) alter levels of serotonin by regulating the transport of serotonin with decreasing the activity of SERT. Therefore, serotonin stays in the synaptic cleft during a longer time. SSRIs are common drug used to treat human behavioural issues. In humans, loss of function in the serotonergic system can lead to different health problems, for instance depression and obsessive compulsive disorder. Currently, this can be treated with the aid of different drugs, including SSRIs. SSRIs increase extracellular levels of serotonin in the brain and help to upregulate serotonin. A commonly used SSRI is fluoxetine, which is the active substance in Prozac™ (Dankoski et al. 2014). Fluoxetine is used worldwide for

4 treating the symptoms for example against depression, attention deficit disorder (ADD), attention deficit hyperactivity disorder (ADHD) and Asperger syndrome (Williams 2010).

Studies are frequently carried out investigating aspects of the serotonergic system in both vertebrates and invertebrates. However, it is still unclear what aspects of the serotonergic system are comparable between these taxa. Hence, this report aims to investigate if the receptors and proteins involved in the

serotonergic system in vertebrates and invertebrates are comparable. This report will mainly focus on humans as a model for vertebrates and insects as a model for invertebrates.

3. Literature review

3.2.1 Biosynthesis and metabolism of serotonin

When reviewing the literature, it becomes clear that the biosynthesis of serotonin is identical in vertebrates and invertebrates (Blenau & Baumann 2001). In vertebrates, the catabolic pathway of serotonin is oxidative deamination by monoamine oxidases. However, little or no activity of monoamine oxidases could be detected in insect nervous tissue, thus the

enzymatic inactivation is probably after amino-terminal tagging (Vleugels et al. 2015).

3.2.2 Receptors involved in the serotonergic system

G-protein-coupled-receptors (GPCR) are receptors found only in eukaryotes and probably 800 genes in human encodes for different types of GPCRs. The

receptors are divided into six classes depending on their functioning. The major class of GPCRs is Class A Rhodopsin-like receptors. The receptors are involved in a lot of different mechanisms in an organism (Bjarnadóttir et al. 2006).

GPCRs in the serotonergic signalling system all fall under the subfamily of rhodopsin-like receptors. All members of this receptor group have their N-terminus located in the extracellular environment and the C-N-terminus located intracellularly. The N-terminal often contains motifs for N-linked glycosylation, and the receptors will be turned off by phosphorylation assisted by serine and threonine located on the C-terminal of the protein (Blenau & Baumann 2001).

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3.2.3 Serotonin receptor subtypes in vertebrates

Receptors in the serotonergic system in vertebrates are both GPCRs and ligand-gated ion-channel receptors (Peroutka & Howell 1994). Six of the seven classes of serotonin receptors belong to the GPCR and one is a ligand-gated

ion-channel. The seven classes of the receptor are named 5-HT1-7. They are

separated into special groups due to their structure and operation in the

serotonergic system. There are also different subtypes of the 5-HT1-7 receptors.

There are presumably a lot of different signalling capabilities due to numbers of posttranslational modifications of the protein-linked receptors (Hoyer et al. 2001). The meaning of posttranslational modifications is that either the N- or C-terminal is altered, for example, by glycosylation, phosphorylation or by

modifying the existing functional group (Swedish MeSH 2017). Studying the GPCRs, five of the receptors inhibit cAMP production, three induce cAMP, three lead to higher concentration of Ca2+ _{and the two others are yet unknown}

(Blenau & Baumann 2001).

The receptor 5-HT1 has five subtypes, named A-F. The receptors 5-HT1A, B and

5-HT D have the same functions in various tissues in different species (Hoyer et

al. 2001). The invertebrate and vertebrate systems probably originate from the same main receptor class (Peroutka & Howell 1994; Hauser et al. 2006).

3.2.4 Serotonin receptor subtypes in invertebrates

Insects’ serotonin receptors are classified with help from vertebrate sequences. Most of the receptors that are found in vertebrates are also found in invertebrates and signalling is similar between the two taxa (Blenau & Baumann 2001). The GPCRs share a high level of sequence homology across taxa (Brody & Cravchik 2000). Serotonergic receptors are found frequently in the brain and ventral nerve cord (Cai et al. 2010). Due to the conserved amino acid sequences and activation of second messengers the serotonergic receptors in insects have been classified as 5-HT1, 5-HT2 and 5-HT7 of the type GPCRs (Table 1). In fruit fly and field

cricket (Gryllus bimaculatus) five different receptors have been found, two subtypes of 5-HT1, two of 5-HT2 and one of 5-HT7. In honey bee (Apis

melllifera) one subtype of 5-HT1 and two of 5-HT2 have been characterized,

while in locust (Locusta migratoria manilensis) and American cockroach

(Periplaneta americana), only one subtype of 5-HT1 and one of 5-HT2 receptors

are found. However, the 5-HT7 receptor is found in all species (Vleugels et al.

2015). Studying the homology in the 5-HT7 receptor between human and fruit

fly showns that the sequences are identical to 39 % and the query coverage is 68 %. Comparing the homology in the 5-HT1 receptor between human and fruit fly

results in 39 % identical and with 89 % query coverage. It is also shown that comparing 5-HT2A between the two species, the ident is 36 % and 5-HT2B results

6 in 29 % identical. (BLAST; UniProt 2017). Noticeable is that subtypes 5-HT3-6

that exists in human seem to lack homologous genes in insects. This indicates that the gene set-up between insects and human is not identical and possess a different number of genes encoding for serotonin receptors.

Table 1. Summary of the number of different subtypes of serotonin receptors in different insect species

Species 5-HT1 5-HT2 5-HT7 Fruit fly 2 2 1 Field cricket 2 2 1 Honey bee 1 2 1 Locust 1 1 1 American cockroach 1 1 1

Analysing the different subtypes of serotonergic receptors implies that subtypes differs between species. Insects possess a 5-HT7 receptor but the number of

subtypes of 5-HT1 and 5-HT2, differs. In vertebrates, the mechanisms for

creating subtypes of receptors seem to be alternative splicing and so does it for invertebrates as well. However, the pharmacological effects are different

between vertebrate and invertebrate receptors. It has been shown that synthetic agonists for human serotonergic receptors do not have the same potency and efficiency on insects’ serotonin receptors (Vleugels et al. 2015).

3.2.5 Serotonin transporter proteins

There are several proteins that are involved in the serotonergic system. One of the most important is the serotonin transporter (SERT or 5-HTT, Uniprot 2017). SERT is a major target for drugs involved in the serotonergic system due to its important function in the system. The transporter is located on the dendrites of nerve cells. It is a part of the sodium-dependent serotonin transporter family and encoded by the SLC6A4 gene. The gene is found on chromosome 17 in humans (Nakamura et al. 2000). The gene that encodes for SERT consists of 14 exons (Murphy & Moya 2011). The transporter transports serotonin from the synaptic cleft to the presynaptic neuron so that it can be re-used. The protein consists of 630 amino acids and is a membrane protein that penetrates the membrane 12 times (Uniprot 2017).

7 Figure 3. X-ray picture of serotonin transporter protein and how it is located in the cell membrane. Red = outer membrane, blue = inner membrane (Protein Data Bank, 2017).

3.2.6 Serotonin transporter protein in vertebrates and invertebrates

The affinity (KM-value) of serotonin for SERT in fruit flies is in the range of

mammalian SERT values (Murphy et al. 2004). However, the transporter has changed somewhat over evolutionary time and the similarities of the gene encoding for SERT are around 50 % when comparing human and fruit fly (Figure 5, Murphy et al. 2004).

Figure 5. Evolutionary tree of the serotonin transporter protein, SERT (Murphy et al. 2004).

Human SERT is quite similar to various other vertebrate species, but the percentage of homology is reduced in comparison to invertebrates (Table 2, Murphy et al. 2004).

8 Table 2. Percentage of homology when comparing different species to the human serotonin transporter protein (Murphy et al. 2004).

Species Homology Human 98 % Rhesus Monkey 93 % Cow 93 % Sheep 90 % Guinea pig 91 % Rat 91 % Mouse 91 % Drosophila 51 % Tobacco hawkmoth 53 % Caenorhabditis elegans 44 % Streptococcus thermophilus 21 %

3.2.7 Blocking the serotoninergic system

Blocking the serotonergic system can be achieved using SSRIs. SSRIs

commonly require 3-6 weeks for reaching observable effects. The efficacy of SSRIs in treating depression and obsessive compulsive disorders is linked to other factors, such as ongoing stress during the treatment (Dankoski et al. 2014). All SSRIs have a flat drug-response curve meaning that there is no reason to exceed the effective minimum concentration of the drug (Preskorn et al. 2004).

3.2.8 Fluoxetine

For the experimental part of this study (see Material and Methods below), fluoxetine (Figure 3) was chosen as an SSRI due to its known effect on humans where it is commonly used as an anti-depressant (FASS 2017). Because human pharmaceuticals enter our waste water, animals are also exposed to this drug (Brodin et al. 2013). Fluoxetine affects the serotonin transporter gene SLC6A4. However, the response of fluoxetine on SERT is related to the allelic variation of the gene (Altar et al. 2013).

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4. Experiment

In addition to my literature review, I took part in an experimental study

investigating the effect of the SSRI fluoxetine in Mediterranean field crickets.

4.1 Material and Method

4.1.1 Subjects

Sexually mature male Mediterranean field crickets (Gryllus bimactulatus) were purchased at the local pet shop (n = 72). Crickets were individually housed in containers (9 x 16 x 10.5 cm) in a laboratory at Linköping University. The containers were covered with a plastic lid and each container had a paper towel covering the bottom. Housing containers contained a cylindrical cardboard piece (as a shelter), food and water, apple slices and agar pieces. Crickets were housed at a temperature of 23 ± 2 °C, with a photoperiod of 12:12 light:dark.

4.1.2 Treatment

Based on previous studies on fall armyworm (Spodoptera frugiperda, Howarth et al. 2002), the concentration of fluoxetine was diluted to yield a final

concentration of 10 µM (see appendix). The dilution was made in phosphate buffered saline (PBS). The buffer was chosen because it matched the

physiological extracellular medium in the cricket. The treated crickets (n = 36) were injected with a volume of 10 µL fluoxetine into the abdominal cavity, with a micro-syringe (Hamilton, Sigma-Aldrich). Control crickets (n = 36) were injected with 10 µL PBS, as a sham injection. The control crickets were handled and injected with pure PBS buffer to exclude that stress could be a confounding factor. Only the injector knew which cricket belonged to which group, to avoid biases.

4.1.3 Exploration

Based on previous work, the experiment started approximately 60 minutes after the injection. This was to have time for fluoxetine to work, but not too long so that the effect would wear away. The specific time was chosen due to previous studies injecting 5-HTP in crickets (Dyakonova & Krushinsky 2012).

Exploration in a novel environment for the crickets was scored by measuring the cricket’s total movement in centimetres (recorded by the program Ethovision) for 15 minutes. Crickets were moved from their home container to a novel container (36 x 21.5 x 22 cm), the bottom of the container was covered with

10 white sand. They were moved from their home container with the help of their home shelter (a cylindrical cardboard piece). To optimize the video tracking, the arenas were empty except from the shelters.

4.1.4 Statistics

Because the data were non-normally distributed, non-parametric statistics were used. To investigate the difference between the experimental and control group in distance explored, a Mann-Whitney U test was used. The data program used for analysing the data was SPSS.

4. 2 Results

Crickets treated with fluoxetine explored significantly less than control crickets (treated crickets, 36.04 ± 16.12 cm, control crickets, 219.08 ± 48.90 cm, U = 386, P = 0.001, Figure 5).

Figure 5. Fluoxetine reduced exploration in male Mediterranean field crickets. Differences in distance explored (given in cm) was greater for control (white) vs experimental (blue) crickets, in a novel arena. Control crickets were injected with phosphate buffer, experimental crickets were injected with fluoxetine. Mean ± SE is given.

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5. General Discussion

My review of the literature indicates that the serotonergic system possess similar, but not identical, receptors in vertebrates and invertebrates. It does however differ in the amount of different subtypes of receptors between the two taxa. The serotonin transporter, SERT, is somewhat different between

vertebrates and invertebrates.

Our experiment showed that fluoxetine decreases the willingness for crickets to explore relative to crickets not exposed to fluoxetine. This result contrasts with a previous study which showed that crickets injected with 5-HTP increased their willingness to fight (Dyakonova & Krushinsky 2012). The different results with comparing the fluoxetine and 5-HTP studies could be explained by fluoxetine being a drug aimed for human receptors and proteins, while 5-HTP exists in both human and insect. In humans, fluoxetine increases serotonin levels and thereby increases the activity of serotonin, which leads to individuals feeling happier and more confident (Vårdguiden 2017). If the feeling of self-confidence increases, it could increase the willingness for a human to explore new surroundings. The serotonin transporter protein is less than ~50 % similar in humans and insects, thus chemicals manipulating transportation of serotonin may not have the same effect in the two taxa. The 5-HT7 receptor is only 39 %

similar between fruit fly and human, but there are more receptors involved in the serotonergic system than transporter proteins, so there could be that another receptor reacts differently with fluoxetine. Another difference between our study and human results is that in humans, SSRIs could take 3-6 weeks to have an effect so more comparable results may be obtained by increasing the cricket exposure time period to 3-6 weeks.

5.1 Social and ethical aspects

The social aspect of this project is that we gained information about how serotonin affects the behaviour in crickets. This could give further information on how the serotonergic system works. Likely we will learn transferable

implications to a range of species, due to that we will learn about the role of serotonin on behaviour. From the experimental study we might also learn about if the functions of receptors and transporters are equal between species and if there is a possibility to do experiments on, for example crickets instead of humans or other mammals on the serotonergic system.

Despite invertebrates not requiring special ethical permits, it is still taken in consideration that they are animals and need to be handled with care and without adding extra stress.

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5.2 Conclusion

The serotonergic systems in vertebrates and invertebrates are somewhat similar in terms of receptors. However, the receptors in the two taxa may not be

influenced by the same SSRIs because important transporters, such as SERT because it seems to differ between species. To investigate this further, I suggest amplifying the transport protein SERT from both vertebrates and invertebrates and trying different methods to investigate the affinity of the transporter with serotonin. This could give insight into whether SERT is different between vertebrates and invertebrates.

With the knowledge of the role of receptors and transporters involved in the serotonergic system, I suggest that using insects as a model organism for research about the human serotonergic system is not appropriate for now because of the differences between the two taxa.

Acknowledgement

Special thanks to my supervisors Hanne Løvlie, Robin Abbey-Lee and Emily Uhrig for all the help they provided. Also a big thank you to the other students Simon Aksoy-Björklund, Kristoffer Lundgren Louise Franzen. Laura Garnham, Josefina Zidar, Sarah Child for feedback and support and for carrying out the experiments.

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References

Altar A, Hornberger J, Shewade A, Cruz V, Garrison J, Mrazek D. (2013). Clinical validity of cytochrome P450 metabolism and serotonin gene variants in psychiatric pharmacotherapy. International Review of Psychiatry. 25. 509-533. Blenau W, Baumann A. (2001). Molecular and Pharmacological Properties of Insect Biogenic Amine Receptors: lessons From Drosophila melanogaster and Apis mellifera. Insect Biochemistry and Physiology. 48. 13-38.

Bjarnadóttir TK, Gloriam DE, Hellstrand SH, Kristiansson H, Fredriksson R, Schiöth HB (2006). Comprehensive repertoire and phylogenetic analysis of the G protein-coupled receptors in human and mouse. Genomics. 88.263–273. BLAST. (2017). https://blast.ncbi.nlm.nih.gov/Blast.cgi (Accessed: 2017-06-16) Brodin T, Fick J, Jonsson M, Klaminder J. (2013). Dilute concentrations of a psychiatric drug alter behavior of fish from natural populations. Science. 339. 814-815.

Brody T, Cravchik A. (2000). Drosophila melanogaster G protein coupled receptors. The Journal of Cell Biology. 150. 83-88

Cai M, Li Z, Fan F, Huang Q, Shao X, Song G. (2010). Design and synthesis of novel insecticides based on the serotonergic ligand 1-[(4-aminophenyl) ethyl]-4-[3-(trifluoromethyl) phenyl] piperazine (PAPP). Journal of Agricultural and

Food Chemistry. 58. 2624-2629.

Corey J, Quick M, Davidson N, Lester H, Guastella J. (1994). A cocaine-sensitive Drosophila serotonin transporter: Cloning, expression, and

electrophysiological characterization. Proceedings of the National Academy of

Science. 91. 1188-1192.

Dierick HA, Greenspan RJ. (2007). Serotonin and neuropeptide F have opposite modulatory effects on fly aggression. Nature Genetics. 39. 678-682.

Dankoski EC, Agster AL, Fox ME, Moy SS, Wightman M. (2014). Facilitation of Serotonin Signalling by SSRIs is Attenuated by Social Isolation.

Neuropsychopharmacology. 39. 2928-2937.

Drugs.com (2017). https://www.drugs.com/monograph/fluoxetine-hydrochloride.html (Accessed: 2017-04-23).

Dyakonova VE, Krushinsky AL. (2012). Serotonin precursor

(5-hydroxytryptophan) causes substantial changes in the fighting behaviour of male crickets, Gryllus bimactulatus. Journal of Comparative Physiology A. 199. 601-609.

14 FASS (Pharmaceutical Specialities in Sweden). (2017).

https://www.fass.se/LIF/product?userType=2&nplId=20090409000071&docTy pe=7#product-information (Accessed: 2017-05-10)

Feyereisen R. (1999). Insect P450 enzymes. Annual Review of Entomology. 1. 507-527.

Hauser F, Cazzamali G, Williamson M, Blenau W, Grimmelikhuijzen CJP. (2006). A review of neurohormone GPCRs present in the fruitfly Drosophila melanogaster and the honey bee Apis mellifera. Progress in Neurobiology. 80. 1-19.

Howarth CJ, Prince RI, Dyker H, Losel PM, Seinsche A, Osborne RH. (2002). Pharmacological characterisation of 5-hydroxytryptamine-induces contractile effects in the isolated gut of the lepidopteran caterpillar Spodoptera frugiperda.

Journal of Insect Physiology. 48. 43-52.

Swedish MeSH, Karolinska Institutet. (2017).

https://mesh.kib.ki.se/term/D011499/protein-processing-post-translational (Accessed 2017-06-01)

Kroeze WK, Kristiansen K, Roth BL. (2002). Molecular biology of serotonin receptors: structure and function at the molecular level. Current Topics in

Medicinal Chemistry. 2. 507–528.

Murphy D, Moya P. (2011). Human Serotonin Transporter Gene (SLC6A4) Variants: Their Contributions to Understanding Pharamcogenmomic and Other Functional G x G and G x E Differences in Health and Disease. Current Opinion

in Pharmacology. 11. 3-10.

Murphy D, Lerner A, Rudnick G, Lesch K-P. (2004). Serotonin

Transporter: Gene, Genetic Disorders, and Pharmacogenetics. Molecular

Interventions. 2. 109-123.

Nakamura M, Ueno S, Sano A, Tanabe H (2000). The human serotonin transporter gene linked polymorphism (5-HTTLPR) shows ten novel allelic variants. Molecular Psychiatry. 5. 32–38.

Nakayama S, Sasaki K. M, Lewis Z, Takahisa M (2012). Dopaminergic system as the mechanism underlying personality in a beetle. Journal of Insect

Physiology. 58. 750-755.

Nelson DR, Koymans L, Katataki T, Stegeman JJ, Feyereisen R, Waxman DJ, Waterman MR, Gotho O, Coon MJ, Estabrook RW, Gunsalus IC, Nebert DW. (1996). P450 superfamily: Update on new sequences, gene mapping, accession numbers and nomenclature. Pharmacogenetics 1. 1-42.

15 Mandrioli R, Forti GC, Raggi MA. (2006). Fluoxetine metabolism and

pharmacological interactions: the role of cytochrome P450. Currently Drug

Metabolism. 2. 127-133.

Peroutka SJ, Howell TA. (1994). The molecular evolution of G protein-coupled receptors: focus on 5-hydroxytryptamine receptors. Neuropharmacology. 33. 319-324.

Preskorn SH, Ross R, Stanga CY. (2004). Selective Serotonin Reuptake Inhibitors. Springer Berlin Heidelberg.

Sitaraman D, Laferriere H, Birman S, Zars T. (2012). Serotonin is critical for rewarded olfactory short-term memory in Drosophila. Journal of Neurogenetics. 26. 238-244.

Strüder HK, Weicker H. (2001). Physiology and pathophysiology of the serotonergic system and its implications on mental and physical performance. Part I. International Journal of Sports-Medicine. 7. 467-481.

Swallow JG, Bubak AN, Grace JL. (2016). The role of monoamines in modulation behaviour. Current Zoology. 62. 253-255.

Tierney AJ. (2001). Structure and function of invertebrate 5-HT receptors: a review. Comparative Biochemistry and Physiology Part A. 128. 791-804. Uniprot. (2017). http://www.uniprot.org/uniprot/P31645 (Accessed 2017-05-01).

Vleugels R, Verlinden H, Vanden Broeck J. (2015). Serotonin, serotonin receptors and their actions in insects. Neurotransmitter. 2. 1.

Vårdguiden. (2017). https://www.1177.se/Jonkopings-lan/Fakta-och-rad/Rad-om-lakemedel/Lakemedel-vid-depression/ (Accessed 2017-06-11).

Yuan Q, Joiner WJ, Sehgal A. (2006). A sleep-promoting role for the Drosophila serotonin receptor 1A. Current Biology. 16. 1051-1062.

Williams K. (2010). Selective serotonin reuptake inhibitors (SSRIs) for autism spectrum disorders (ASD). Cochrane Database of Systematic reviews.8.

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Appendix

Calculations of dilution for fluoxetine

Total weight of fluoxetine = 10 mg Molecular weight= 345, 79 g/mol

5 𝑚𝑔 50 𝑚𝐿⁄ = 0.1 𝑚𝑔 𝑚𝐿⁄ = 0.1 𝑔 𝐿⁄ (1)

0.1

345.79 = 0.000289 𝑚𝑜𝑙 = 289µ𝑀 (2)

289 µ𝑀 ∙ 𝑣₁ = 10 µ𝑀 ∙ 100 𝑚𝐿 (3)

𝑣₁ = 3,46 𝑚𝐿 (4)

From (4) it was known that to obtain a concentration of 10 µM, 3.46 mL of fluoxetine had to be diluted in 96.54 mL PBS.