Even More Fish on Prozac

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EVEN MORE FISH ON PROZAC: INFLUENCE OF THE 5 HT 6 RECEPTOR AND STRESS ON LEARNING AND MEMORY BEHAVIOR IN GOLDFISH (CARASSIS AURATUS) BY KEVIN CHRISTOPHER LAW A Thesis Submitted to the Division of Natural Sciences New College of Florida in part ial fulfillment of the requirements for the degree Bachelor of Arts Under the sponsorship of Dr. Alfred Beulig Sarasota, Florida April, 2011

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ii Acknowledgements I would like to thank my thesis sponsor, Dr. Alfred Beulig for guiding me through the d aunting process of creating this thesis, as well as my committee members, Dr.'s Leo Demski and Gordon Bauer. I would also like to thank Joel Beaver for helping me take care of my subjects and keep them alive. Thanks go out to my parents and my Uncle Richar d and Aunt Jo, without who se financial help I would not have been able to finish my college career. I would also like to thank my girlfriend, Sarah for bearing with me through thick and thin, this year, and the rest of my friends who I have been able to un wind with. At last, I would like to thank all of the goldfish who made this experiment possible.

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iii Table of Contents Title ................................ ................................ ................................ ....................... i Acknowledgements ................................ ................................ ............................ ii Table of Contents ................................ ................................ .............................. iii Abstract ................................ ................................ ................................ ............... v Serotonin: Discovery and Historical Overview ................................ ................ 1 Anatomy of the Serotonin System ................................ ................................ .... 2 Phylogeny of Serotonin ................................ ................................ ..................... 4 Evolutionary History of the Teleosts ................................ ................................ 7 Neuronal Function ................................ ................................ ............................ 10 G Protein Coupled Receptors ................................ ................................ ......... 13 The 5 HT 6 Receptor ................................ ................................ .......................... 14 The Serotonin Reuptake Transporter and Antidepressant Drugs ................ 17 Serotonin Pharmacology ................................ ................................ ................. 18 Fluoxetine ................................ ................................ ................................ ......... 20 SB 258,585 ................................ ................................ ................................ ........ 22 Shuttle Box Avoidance Conditioning ................................ ............................. 23 Rationale for Current Experiment ................................ ................................ ... 25 Materials and Methods ................................ ................................ ..................... 29 Subjects ................................ ................................ ................................ .......... 29 Home Aquaria and Maintenance ................................ ................................ ..... 30 Shuttle Box Aquaria ................................ ................................ ......................... 32 Drug Administration ................................ ................................ ......................... 34 Subject Deaths and Anomalies ................................ ................................ ....... 36 Experimental Protocol ................................ ................................ ..................... 39 Results ................................ ................................ ................................ .............. 40 Avoidance Responses ................................ ................................ ..................... 40 Failure Responses ................................ ................................ .......................... 44 Escape Responses ................................ ................................ ......................... 48 Avoid Latency ................................ ................................ ................................ .. 49 Escape Latency ................................ ................................ ............................... 52 Discussion ................................ ................................ ................................ ........ 53 Performance Variability ................................ ................................ ................... 55 Support of Hypotheses and Conclusions ................................ ........................ 59 Appendix A: Raw Data ................................ ................................ ..................... 62 Avoidance, Escape, and Failure Responses ................................ ................... 62 Avoidance and Escape Latency ................................ ................................ ...... 74 Appendix B: Su bject Deaths and Anomalies ................................ ................. 85 Subject Deaths ................................ ................................ ................................ 85

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iv Other Anomalies ................................ ................................ ............................. 86 Append ix C: Programming Methodology ................................ ....................... 8 7 References ................................ ................................ ................................ ........ 89

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v EVEN MORE FISH ON PROZAC: INFLUENCE OF THE 5 HT 6 RECEPTOR AND STRESS ON LEARNING AND MEMORY BEHAVIOR IN GOLDFIS H (CARASSIS AURATUS) Kevin Law New College of Florida, 2011 ABSTRACT This experiment is a follow up to previous work investigating the conservation of stress mechanisms and serotonergic function in teleosts as compared to other chordates. The effects o f manipulating elements of the serotonin system were tested on goldfish (Carassius auratus), which were subjected to the shuttle box two way avoidance paradigm. After the most recent study failed to find a significant difference in performance between sub jects treated with the antidepressant drug, fluoxetine, mainly due to high variability within subject groups, the experiment was repeated with the same dose with an effort to minimize within group variability. Also, as inhibition of many serotonin receptor s has been found to increase performance in memory based tasks, the selective 5 HT 6 antagonist, SB 258,585 was administered to another group in order to gain evidence that goldfish possess this receptor and that its function is similar to that of mammals. Finally, a group was administered both drugs simultaneously to investigate some of the potential stress related effects of a 5 HT 6 antagonist in the presence of a known stress reducing agent (fluoxetine).

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vi This study is the first in this line of research t o show a statistically significant difference between the performance of goldfish treated with fluoxetine and that of the control. Surprisingly, goldfish treated with SB 258,585 performed more poorly than control although the difference was not found to b e significant. This result suggests that some aspect of the 5 HT 6 receptor is conserved between goldfish and mammals, but that there are one or more fundamental differences in its function. Also, the combined treatment group performed more poorly than the SB group but better than the fluoxetine group, although no significant difference was found between either of the treated groups. These results confirm the conclusions suggested by the previous studies about fluoxetine, as well as revealing a possible diff erence between the serotonin system of teleosts and that of mammals, regarding the 5 HT 6 receptor. Dr. Alfred Beulig Division of Natural Sciences

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1 Serotonin: Discovery and Historical Overview Serotonin was discovered in 1935 by Italian scientist, Vittori o Erspamer. Erspamer was researching the smooth muscle of the gut when he discovered that a hitherto unknown substance, extracted from enterochromaffin cells, caused contractions in the uteri of rats He also found, through further research, that it had a similar effect on the hearts of molluscs and could also be found in the salivary glands of the common octopus ( Octopus vulgaris ) He called the substance enteramine (Whitaker Azmitia 1999) In 1948, American scientists Irvine Page, Arda Green, and Maurice Rapport discovered a chemical in blood serum while searching for vasoconstrictors involved in hypertension. They named this chemical serotonin (Rapport et al. 1948) In 1949, Rapport elucidated the structure of serotonin as 5 hydroxytryptamine ( 5 HT ) (Rapp ort 1949). It was not until 1952 that serotonin and enteramine were determined to be the same chemical (Whitaker Azmitia 1999) Meanwhile, American scientist, Betty Twarog was searching for a neurotransmitter which inhibited muscle catch (the bivalve abil ity to "lock" their shells closed) in the mussel ( Mytilus edulis ). After Erspamer published his research, Twarog tested serotonin for this property (Whitaker Azmitia 1999) This was the first evidence of serotonin as a neurotransmitter. In 1953 Twarog

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2 col laborated with Page and together, they discovered the first evidence of serotonin in the mammalian brain ( Twarog & Page 1953 ). Serotonin was first implicated to be candidate for the study of mental illness by Dr D. W. Woolley, following his research on lys ergic acid diethylamide (LSD) (Whitaker Azmitia 1999) This was the start of an entire field of research into the role of serotonin in everything from depression to schizophrenia. Anatomy of the Serotonin System The serotonergic system originates in the ra phe nuclei in the medial portion of the brainstem. Its projections are highly diverse and spread throughout much of the brain. The system can be subdivided into two major subsystems: the rostral serotonergic system whose nuclei are located in the midbrain and project to the forebrain, and a caudal system whose nuclei are located in the medulla and project to the spinal cord (Tork 1990) The bodies of serotonergic cells of the rostral subdivision are primarily located in the caudal linear nucleus, the dorsa l raphe nucleus, and the median raphe nucleus. The cells of the caudal linear nucleus are small with few dendrites. The dorsal raphe nucleus can be subdivided into 5 subnuclei with varying cell sizes and cell densities, while the median raphe nucleus can b e subdivided into two sections with varying dendrite orientations. Both the dorsal and median raphe nuclei send their projections to the medial forebrain. These

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3 projections terminate in the limbic system, thalamus, hypothalamus, striatum, hippocampus, amyg dala and extensive areas of the cerebral cortex (Tork 1990). Fig. 1: Schematic of serotonergic nuclei and projections of the rostral subdivision. From Tork 2006. Adapted from Stone 1990. The bodies of serotonergic cells in the caudal subdivision are pr imarily located in the raphe magnus nucleus, the raphe pallidus nucleus, and the raphe obscurus nucleus. The axons of these nuclei project into the spinal cord (Tork 1990). This document focuses on the 5 HT 6 receptor. S ince the 5 HT 6 receptor is found exc lusively in the brain, this document will not elaborate on the caudal subdivision.

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4 Phylogenetic History of Serotonin The phylogenetic history of serotonin is complex and has a very long history. Although primarily known as a neurotransmitter in vertebrates it also serves a wide range of other functions in everything from animals (including invertebrates) (Erspamer 1948), to plants (Feldman & Lee 1985) and even bacteria (Oleskin et al. 1998) This section will focus primarily on serotonin's role as a neur ochemical. Serotonin can be found, functioning as a neurochemical, in virtually all organisms with a nervous system. The cniderians and ctenophorans, whose nervous systems are so simple that they lack unidirectional synapses use serotonin as a neurochemi cal Radiolabeling techniques have been used to map the receptor and transporter distribution in the sea pansy, Renilla koellikeri in which it acts as a neurohormone (a chemical messenger which travels throughout parts of the nervous system, rather than b eing localized to a synapse) (Dergham & Anctil 1998) Cubozoan jellyfish, are unique in that they possess complex eyes with lenses. The ganglia connected to these eyes have chemical synapses in which serotonin has been detected (Martin 2004) Although pro gress is being made, large gaps still exist in the literature regarding serotonin in these phyla. The phylogeny of serotonin neurotransmission is somewhat better studied in mollusks, thanks to the position of Aply sia californica as a model organism. Seroto nin acts as a true synaptic neurotransmitter in this species. It has been

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5 found to increase the excitability of sensory neurons, influencing a wide array of behaviors, including feeding, swimming away from threats, and other sensory behaviors (Hammer et al 1989, Cleary et al. 1995 ). Serotonin is also involved in the modulation of synaptic action for the siphon/gill withdrawal reflex in Aplysia (Kandel & Schwartz 1982). As stated previously, serotonin has also been found in the salivary glands of Octopus vu lgaris and relaxes the muscle catch mechanism in Mytilus edulis. The serotonin system has also been well studied in arthropods. The fruit fly ( Drosophila melanogaster ) has long been used by neuroscientists as a model organism. This species has even been su bjected to avoidance tasks designed to test learning and memory performance, similar to that used in this experiment. The results have shown that flies that have been genetically or pharmacologically altered for reduced serotonin have reduced performance i n avoidance tasks associated with place memory (Sitaraman et al. 2008). Serotonin has been best studied as a neurotransmitter in the chordates, particula rly the mammals. Serotonin is found in the brains of all chordates which can reasonably be said to have brains within which it serves a more complex role than in any other phylum. Most serotonin research in chordates has been conducted on rats and mice (Gerard et al. 1997, Loiseau et al. 2007 ) Given that they are mammals, and have brain architecture simil ar to humans, this is understandable. In the cat, immunohistochemical studies have been done to determine the anatomical distribution of serotonergic neurons, and the results

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6 showed that it is very similar to that of humans (see previous section) (Leger et al. 2001). To date, there have been 15 different serotonin receptors identified in humans, divided among 7 major types (5 HT 1 7 ). Given the pace of research in molecular biology, more are likely to be discovered. Although the exact protein sequences diffe r, close analogs of the vast majority of these receptors have been identified in other mammals, notably, rats. The serotonin system is so well studied in rats that the effects of each of the receptors have been carefully studied for a wide range of behavio rs, including cognition (Leger et al. 2001). Until t his current study, the cognition enhancing effects of the 5 HT 6 receptor have been studied exclusively in the rat (see section on the 5 HT 6 receptor for more on this). Considering the relative timeframe o f the evolution of each of these phyla, it is apparent that serotonin's role as a neurochemical becomes consi stently more complex as the complexity of the organism's nervous system increases In the most primitive phyla, it serves a role primarily as a neu rohormone. In mollusks, arthropods, and chordates it functions primarily as a modulatory neurotransmitter (sometimes as a direct neurotransmitter) (Weiger 1997). As well as the serotonin system of mammals has been studied, the phylogeny of serotonin in th e lower chorda tes remains largely incomplete. Among these taxa, the teleosts have been used as model organisms in a variety of neurobiological studies. The zebrafish ( Danio rerio ) and the goldfish

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7 ( Carassius auratus ) both t eleosts of the family Cyprinidae are the best studied of these. The distribution of serotonin in the teleost brain has been well documented. Using immunoreactive serotonin, serotonergic neurons have been identified in the hypothalamus, nucleus dorsolateralis thalami, projecting to the ro stral midbrain tegmentum, as well as between the fibers of the fasciculus longitudinalis medialis and in the pituitary gland and parts of the medulla oblongata (Kah & Chambolle 1983) Although genetic homologues of many of the serotonin receptors have bee n identified in these species, it is currently unknown whether or not a functional equivalent of the 5 HT 6 receptor exists in either. Evolutionary History of the Teleosts The teleosts are an infraclass which comprises the most phylogenetically advanced bon y fish (class: O steichthyes). They belong to the subclass, A ctinopterygii, the ray finned fishes, and comprise more than half of the known species of vertebrates. The evolution of fish begins with the evolution of the first vertebrates: the agnathans. The agnathans are colloquially known as "jawless fish," and are represented tod ay by only the hagfish (class: M yxin oidea) and the lamprey (class: P etromyzontida). Agnathans lack the jaws and paired fins which have come to characterize modern fish groups. This group evolved during the early Ordovician period 470 480 million years ago making the oldest organisms that could reasonably be called "fish" (Erdtmann et al. 2000). Although only the

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8 hagfish and lamprey remain today, in ancient times there were a great deal more species many of which possessed bon y armor over much of thei r bodies and bony shields on their heads known as O st racoderms. One such group, the O steostraci, which first appeared in the Ordovician and reached their evolutionary peak (and subsequ ently, their extinction) during the Devonian period, 416 360 million years ago may have given rise to the jawed fishes (Long, 1996). The first jawed fishes a ppeared in the late Ordovician, and were represented by two classes: Acanthodii and the P lacodermi The placoderms resembled the osteostracans, with their bony armor except for their fully developed jaws. The acanthodians lacked the head shields but had bony scales over their bodies. Although they had cartilaginous skeletons, the presence of otoliths in their inner ears imply that they may have shared a close common ancestor to osteichthyes (Miles, 1965) The osteichthyans first appeared in the late Silurian period (around 410 million years ago), but their great diversification did not occur until the Devonian. The main defining characteristic is an internal skeleton made of bone. The osteichthyans are di vided into two subclasses: the A ctinopterygii and the sarcopterygii. The Sarcopterygii or fleshy finned fishes, are represented today by only a few sp ecies, including the lungfishes and the coelacanth. They are characterized by four paired fins containing bone and muscle, similar to limbs. Due to recent discoveries of so called "fishapods" such as T iktaalik, it is believed

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9 that the sarcopterygians gave rise to the tetrapods, to which all terrestrial vertebrates belong (Shubin et al. 2006) The actinopterygians, or ray finned fishes, have paired fins composed of a membranous tissue stretched across stiff spines (lepidotrichia). These spines are anchored d irectly to the pectoral and pelvic girdles. The actinopterygians are s ubdivided into 4 infraclasses: Polypteri, Chondrostei, Holostei, and T eleostei. The Polypteri include a single group called the reed fish or bichir. Chondrostei is the next mo st primitiv e clade and contain the sturgeon, and paddlefish. They are characterized by a primarily cartilaginous skeleton which is only partially ossified and a heterocercal tail The Holostei have fully ossified skeletons but a primitive jaw structure and somewhat h eterocercal tails and comprise only the gars and bowfins (Long 1996) By far the most diverse and advanced group of bony fishes are the T eleosts, comprising some 20,000 known species in around 40 orders. Teleosts first appeared during the Triassic period around 250 million years ago. They are characterized by a homocercal tail and an articulated maxilla and premaxilla which allow them to protrude their jaws outward from their mouths (Long, 1996) Among the T eleosts goldfish belong to the order, C yprinif ormes, which includes the loaches, carps, and minnows. Within this ord er, they belong to the family, C yprinidae, which includes the carps (including goldfish) and minnows (Long, 1996)

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10 Goldfish are commonly used as a model organism in neuroscience due to t heir brains and behaviors bein g complex enough to represent an advanced vertebrate while being much easier to breed and maintain (and less expensive) than mammals. Furthermore, when testing chemicals on them, if the chemical in question is water soluble a nd can be absorbed through the gills it can be administered simply by adding it to their aquarium water Also, many chemicals which do not cross the blood brain barrier in mammals, readily cross it in teleost fish. Serotonin is one such chemical (Genot et al. 1981). Neuronal Function Nerve cells, or neurons, are electrochemical systems. Within a single neuron, an electrical current (or action potential) is propagated via the movement of ions through channels in the cell membrane. Like most cells, neurons have a membrane potential of around 70 mV. This is the result of the distribution of sodium and potassium ions inside and outside the cell. Active transport via ATP powered, sodium/potassium pumps establishes the ion concentration gradient by pumping 2 po tassium ions into the cell, while pumping 3 sodium ions out of the cell. This way, the inside of the cell is more negat ively charged than the outside (Koksoy 2002) The membrane of a neuron is lined with voltage gated sodium and potassium channels. These c hannels are sensitive to increases in the membrane potential, known as depolarization s. When the membrane potential de creases to approximately 55 mV, the sodium channels open. Sodium rushes down its concentration gradient, into the cell, raising the membr ane potential to

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11 around +40 mV. Once the potential reaches a certain point, the sodium channels inactivate and the potassium channels open, causing potassium to rush out of the cell, thus dropping the potential, roughly, back to its original value. All of this occurs within approximately one mil lisecond (Hodgkin & Huxley 1952) This system is effective for a signal traveling through a single cell, but more complex mechanisms must come into play in order for multiple neurons to communicate with each other. A neuron is composed of three major structures: the soma, the axon(s), and the dendrites. While the soma contains the nucleus and many of the organelles, and provides most of the metabolic function of the neuron it is the axons and dendrites (and, in part, the soma) which are involved in interneuron communication. An axon from one neuron interfaces with a dendrite from another neuron across a tiny gap called the synaptic cleft or synapse. At the tip of an axon (the axon terminal), there is a collection of t iny, membrane bound vesicles containing small molecules called neurotransmitters. The neurotransmitters, such as serotonin, are synthesized in the soma, and then packaged into vesicles in the golgi complex. They are then trafficked down the axons along mi crotubules via a motor protein called kinesin (Vale et al. 1985). Once at the axon terminal, the neurotransmitters are absorbed by synaptic vesicles. These vesicles are also formed in the golgi complex, but they become bound to the cytoskeleton via protein s called synapsins after they are trafficked down to the axon terminal (Lyn & Scheller 2000).

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12 When the action potential arrives at the axon terminal, it opens voltage gated calcium channels. The influx of calcium activiates calcium/calmodulin kinase II (Ca MKII) which phosphorylates the synapsins, causing them to unbind the synaptic vesicles, allowing these vesicles to mobilize. Once mobilized, the vesicles must dock with the synaptic membrane. This is mediated by proteins anchored to the vesicle membrane wh ich interact with proteins anchored to the synaptic membrane called SNAREs. The SNAREs form a complex which holds the vesicle in place. Next, the SNARE complex is "primed" by the action of the ATPase NSF. This leads to the final step in which calcium initi ates a fusion between the vesicle membrane and the synaptic membrane, thus releasing the neurotransmitter molecules into the synaptic cleft. This step is not fully understood with regards to how calcium interacts with the SNARE complex, causing fusion (Lyn and Scheller 2000). Once the neurotransmitter is released into the synaptic cleft, it must interact with the post synaptic neuron. This is achieved by binding to receptors embedded in the plasma membrane of the post synaptic cell. There are a wide range o f different types of receptors: there are 7 for serotonin alone and among those, there are a number of sub types. However, virtually all receptors can be divided into two major categories: ionotropic and metabotropic. Ionotropic receptors are one type of ligand gated receptor or ion channel (LGICs) This type of receptor has the most direct mode of action They are simply binding sites on ion channels that, when neurotransmitter molecules

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13 attach, cause a conformational change in channel structure such that the channel becomes open to the passage of ions Beyond this, they function like any other ion channel ions pass through them down their concentration gradients and alter the membrane potential. Among the serotonin receptors, only the 5 HT 3 receptor is a LGIC (Maricq et al. 1991) G Protein Coupled Receptors The remaining serotonin receptors belong to a superfamily of proteins called G protein coupled receptors (GPCRs). These are the metabotropic receptors. Like LGICs, GPCRs are activated by neurotransmitt er chemicals released into the synapse by the pre synaptic neuron. From here, the mode of action between the two differs. As the name implies, the receptor is coupled to a specific G protein. Structurally, they consist of 7 transmembrane domains, 3 intrace llular loops, 3 extracellular loops, an intracellular C terminus, and a n extracellular N terminus. They also include a ligand binding site and a G protein binding site. There are several types of G proteins, each with unique functions. A G protein is a he terotrimeric complex comprised of a n a and a subunit. The and subunits form a complex and are usually anchored to the cell membrane near the receptor. This complex does not typically serve a role beyond an anchoring point for the subunit, but there are cases in which it can act as a signaling molecule itself. When a ligand binds to the GPCR, it induces a

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14 conformational change which allows it to activate the subunit of the G protein by exchanging its bound GDP (guanosine diphosphate) with a m olecule of GTP (guanosine triphosphate). Once this exchange takes place, the subunit dissociates from the complex and becomes an in tracellular signaling molecule (Neves et al. 2002) There are several different types of G subunits each with a uniqu e function One of these is the G s subunit (Monsma et al. 1993) Once activated, the G s subunit binds to, and activates adenylate cyclase (AC) This enzyme synthesizes cyclic adenosine monophosphate (cAMP) from adenosine triphosphate (ATP). Then, cAMP ca n, in turn activate other pathways in the cell. There are LGICs which are not located at the synapse, but are instead activated internally, by cAMP (Pedarzani & Storm 1995) cAMP can also activate protein kinase A (PKA), an enzyme which phosphorylates seri ne and threonine residues of a wide range of proteins. In neurons, PKA can phosphorylate a certain type of ion channel (Neves et al. 2002) The 5 HT 6 Receptor The 5 HT 6 receptor was discovered simultaneously by two separate parties in 1993 using PCR and ge ne sequencing techniques on rat cells (Monsma et al. 1993 Ruat et al. 1993 ). It was identified as a member of the superfamily of G protein coupled receptors and shared significant portions of its gene sequence with the many of the serotonin receptors. It was found that in

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15 cells transfected with this receptor, serotonin strongly increased activity of AC indicating that the 5 HT 6 receptor is coupled to a G protein with the G s subunit (Monsma et al. 1993). There are several sub types of AC which are activated by different mechanisms. G s from the 5 HT 6 receptor activates type 5 AC. Interestingly, the 5 HT 7a receptor, which is also coupled to G s has been found to activate type 1 and type 8 AC, which are calcium/calmodulin sensitive (Baker et al. 1998). Gene sequencing has revealed that the gene for the 5 HT 6 receptor is mapped to the human chromosome region 1p35 p36 (Kohen et al. 1996). The gene codes for a 440 residue polypeptid e. This polypeptide sequence is an 89% match to the 5 HT 6 receptor in the rat. Immuno localization techniques have been used to determine the distribution of the 5 HT 6 receptor. 5 HT 6 like immunoreactive material was found in significant amounts in the ple xiform layer of the olfactory tubercle, frontal and entorhinal areas of the cerebral cortex, nucleus accumbens, striatum, and molecular layer of the cerebellum, as well as several areas of the hippocampus including the strata oriens, radiatum of the CA1 ar ea, and molecular layer of the dentate gyrus (Gerard et al. 1997). This was consistent with previous studies which used techniques such as reverse transcription followed by polymerase chain reaction (PCR)(Gerard et al. 1996) to determine the distribution o f 5 HT 6 mRNA. The presence of mRNA in close proximity to the receptors they code for

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16 indicate that the receptors are placed very close to where they are synthesized (Gerard et al. 1997). These structures, especially the hippocampus, are strongly correlated with spatial or episodic memory formation. Inhibition of the 5 HT 6 receptor is believed to increase glutamate and acetylcholine production in the hippocampus, which is strongly correlated with an increased ability to encode spatial memory (Mitchell & Neum aier 2005). T he rat is currently the only animal on which the pro cognitive effects of blocking the 5 HT 6 receptor have been studied. The 5 HT 6 antagonist, SB 271,046 caused increased performance in the shuttle box two way avoidance paradigm in rats (Dawso n et al. 2001). Other antagonists have shown slightly increased spatial memory performance in the Morris water maze in rats (Woolley et al. 2001). Interestingly, acute administration of these drugs had effects only on memory retention and not acquisition i n these tasks. Studies in which the drugs were administered chronically, however, produced significant positive results with regard to memory acquisition and retention indicating that the receptor is involved with neuroplasticity or long term potentiation (LTP) (Fone, 2008) Considering the anatomical distribution of the 5 HT 6 receptor, coupled with aforesaid work early research strongly implied that this receptor affects memory far mor e than stress levels. This would make it a valuable target for cogniti on and memory enhancement in patients with neurological disorders.

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17 Even more intriguing is research which indicates that antagonists of this receptor actually provide an anxiolytic or antidepressant effect in rats, rather than inhibiting such effects (Wes oloska et al. 2007). Furthermore, research in rats using SB 271,046 (5 HT 6 antagonist) and the serotonin and norepinephrine reuptake inhibitor antidepressants, desipramine and venlafaxine, have shown that the two drugs have a contradictory effect on each o ther. The nootropic (cognition boosting) effects of SB were reduced by the antidepressants, and the anxiolytic effects of the antidepressants were reduced by the SB (Dawson & Li 2003). This would imply that co administration of a 5 HT 6 antagonist with an a nxiolytic would cause the two to cancel each other out. The Serotonin Reuptake Transporter and Antidepressant Drugs After serotonin (or any neurotransmitter) is released into the synapse and binds to receptors, it must quickly dissociate from the receptors This and all excess serotonin must then be rapidly removed from the synaptic cleft, in order to reset the synapse. This is accomplished by serotonin reuptake transporters (SERTs) embedded in the plasma membrane of the pre synaptic neuron. These are 12 tr ansmembrane domain proteins which bind serotonin and shuttle it from the synaptic cleft into the cytoplasm of the pre synaptic cell (Barker & Blakely 1995) From here, two thing s can occur. The most common is breakdown of serotonin by the enzyme, monoamine oxidase (MAO). The products of this reaction are then packed into vesicles and transported back to the soma for recycling. However, the serotonin can also be directly rec ycled at the axon

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18 terminal, by SERTs embedded in the membranes of synaptic vesicles. Since a serotonin deficiency has been correlated with depression, both SERTs and MAO have been the target of antidepressant drugs. The first antidepressant drugs were MAO inhibitors (MAOIs). By preventing the breakdown of serotonin at the axon terminal, mo re serotonin is left available, there. However, MAO not only catalyzes the breakdown of serotonin, but a wide range of other neurotransmitters, including dopamine, and norepiniphrine. This, combined with disruption of the natural metabolism of monoamines, caused many negative and sometimes, permanent side effects. As a result, MAOIs are now used only in cases of extreme or atypical depression (Lenox & Frazier 2002) In the 1970s, a new class of antidepressant was discovered: the selective serotonin reuptake inhibitor (SSRI). This class of drug works by blocking the action of the SERTs, causing serotonin to remain in the synapse longer, in the hope of increasing the number of receptors which bind it with each vesicle release d Today SSRIs are widely used arou nd the world. One of the most common and potent is fluoxetine hydrochloride (Prozac) (Wong et al. 1995) Serotonin Pharmacology Serotonin is one of the best studied of all neurotransmitters and its effects have been found to be highly varied. Serotonin i s the common name for 5 hydroxytryptamine (5 HT). It has a molecular weight of 176.09. It is

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19 biosynthesized from the amino acid L tryptophan. Dietery L tryptophan is hydroxylated by the enzyme, tryptophan hydroxylase to 5 hydroxytryptophan (5 HTP). Due to naturally low systemic concentrations of tryptophan, this is the rate limiting step of the biosynthesis (Fenstrom & Wurtman 1971). Then, 5 HTP is decarboxylated by the enzyme, aromatic L amino acid decarboxylase, yielding 5 HT (serotonin). Fig. 2: Ser otoni n (5 hydroxytryptamine) molecular structure. From IUPHAR Database. Although primarily considered a CNS neurotransmitter, serotonin is also found in a variety of organ systems. It can act as a vasoconstrictor or dilator, affects heart rate, is involved in respiratory regulation, affects the urinary tract, the reproductive organs, mammary glands, and perhaps most drastically, regulates most systems in the digestive tract (Berger et al. 2008). In fact, roughly

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20 80% of the serotonin in the body is found in the gut, which is evidenced by its effects on feeding behavior in most organisms. However, the focus of this document is on serotonin's effects in the CNS. Because the serotonergic system projects to so many different parts of the brain, it has an influenc e on virtually every aspect of behavior. Its effects have been studied by observing behavior in subjects deficient in serotonin and include appetite, aggression, sleep/wake cycles, mood, and cognition (Airan et al. 2007, Canli & Lesch 2007, Roth et al. 200 3). Of particular interest to this study is serotonin's role in both cognition and mood. Based on the success of SSRIs in treating symptoms of depression serotonin is believed to have an anxiolytic effect: providing subjects with a sense of contentment an d well being. It has also been established that blocking certain serotonin receptors increases cognitive performance in learning based tasks for acquisition, consolidation, and retrieval of memory However, the precise effect of each of the 5 HT receptors is not fully understood. Fluoxetine Fluoxetine hydrochloride ( N methyl 3 phenyl 3 [4 (trifluoromethyl) phenoxy] propan 1 amine) (marketed commercially as Prozac) is one of the most widely used a ntidepressants in the world. It has a molecular mass of 309.33 g/mol and a solubility in water of 10 mmol/L It was traditionally believed to function as an SSRI, inhibiting stress by increasing synaptic serotonin concentration. Recently, however, new research has come to light which implies

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21 that at the doses used in clinical subjects, the increase in synaptic serotonin is negligible. Instead, it has been suggested that fluoxetine exerts its anxiolytic effect by increasing levels of the endogenous neurosteroid, allopregnanolone (Allo). Allo is a powerful upmodulator of the GABA A receptor, which gives it a profile more like a powerful benzodiazapine type anxiolytic. This family of drugs increases activity in some of the brain's major inhibitory centers, and has been used for the treatment of clinical anxiety. This effect appears to be completely independent of fluoxetine's inhibitory effects at the SERT (Pinna et al. 2009) Fig. 3: Fluoxetine ( N methyl 3 phenyl 3 [4 (trifluoromethyl) phenoxy] propan 1 amine ) molecular structure. From IUPHAR Database.

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22 If this is indeed the case, administration of serotonin antagonists in conjunction with fluoxetine should produce an anxiolytic effect regardless of which serotonin receptors are blocked. SB 258,585 Recently a number of chemicals have been discovered which show high affinit y for and high specificity to the 5 HT 6 receptor. Among these is SB 258,585 (4 Iodo N [4 methoxy 3 (4 methyl piperazin 1 yl) phenyl] benzenesulphonamide). This compound has a molecular mass of 487.354 g/mol and a solubility in water of 10 mmol/L. Binding s tudies in human, rat, and pig brain tissue have shown that it has an affinity for the 5 HT 6 receptor nearly 100 fold greater than any other 5 HT receptor known (Hirst et al. 2000) Fig 4: SB 258,585 ( 4 Iodo N [4 methoxy 3 (4 methyl piperazin 1 yl) phenyl ] benzenesulphonamide ) molecular structure. Adapted from Hirst et al. 2000. Since its discovery, other ligands have been discovered with even higher specificity to the 5 HT 6 receptor This has led to SB 258,585 being replaced by such ligands as SB 271,046 (Dawson et al. 2001), SB 357,134 (Stean et al. 2002) and SB 399,885 (Hirst et al. 2006). SB 399,885 is possibly the most

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23 specific, with an affinity for the 5 HT 6 receptor 200 fold higher than any other receptor tested. All of these antagonists show similar results in rats. Chronic administration consistently leads to improved memory, as evidenced by performance in numerous paradigms (see above). SB 258,585 was chosen for this study primarily due to availability. Despite the fact that it is no longer conside red the "best" 5 HT 6 antagonist, it still has high specificity and is well suited to this experiment. Shuttle Box Avoidance Conditioning In order to measure a subject's ability to learn, it is necessary to have a paradigm which can be used to test it. Clas sical conditioning is a paradigm, originally developed in the early 1900s by Ivan Pavlov, which seeks to develop a cognitive association between two unrelated stimuli. Pavlov was researching dogs and realized that they would salivate in the presence of foo d. The food is known as the unconditional stimulus because it naturally elicits a specific behavior (salivation). However, by presenting the food with another, unrelated stimulus, such as ringing a bell, he found that the dogs would learn to associate this "conditional stimulus" with food. Eventually, when the conditional stimulus was presented without the unconditional stimulus, the subject would still produce the behavior (the dogs would salivate) (Todes, 2002)

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24 This paradigm, known as classical condition ing or Pavlovian conditioning, has been widely used in animal behavior research to measure learning and memory performance. Another paradigm which must be employed in t his line of research is called instrumental conditioning (or operant c onditioning). Inst rumental conditioning, which was first investigated in the late 1800s by Edward Lee Thorndike, in which a subject is conditioned to produce a specific behavior on something in its environment, in order to elicit a result that does not initially appear to b e related to the action This result can be either the presentation of a positive stimulus or the cessation of a negative stimulus. For example, Thorndike investigated the ability of cats to learn how to escape from a box by activating a lever which would open the door (Thorndike, 1898). The current experiment utilizes the shuttle box two way avoidance paradigm. This paradigm combines both classical conditioning and instrumental conditioning. The shuttle box is a variant of the operant conditioning chamber (sometimes referred to as the Skinner Box), developed in the early 1930s by B. F. Skinner (Skinner, 1938) As with instrumental conditioning, a subject must produce a specific behavior to remove a seemingly unrelated, negative stimulus. In the case of the shuttle box, this r elationship involves crossing from one side of the chamber to the other in order to escape from a shock. However, an operant conditioning chamber also incorporates classical conditioning by introducing a conditional stimulus. The subject is expected to learn to associate the neutral

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25 conditional stimulus with the negative unconditional stimulus, and begin performing the desired behavioral task during the conditional stimulus in order to completely avoid the unconditional stimulus. See the Materials and Methods section and Appendix C for a detailed description of the shuttle box paradigm as was used in this experiment. Rationale for Current Experiment It is currently unknown whether or not the 5 HT 6 receptor is conserved in goldfish. There h ave not been any studie s to date designed to confirm whether or not goldfish have a 5 HT 6 receptor. Genetic research on zebrafish has turned up the existence of a hypothetical protein which is a 54% match to the human 5 HT 6 receptor (Best & Alderton 2008). This is not a particularly close match and the gene was found in zebrafish, not goldfish. However, both species belong to the family cyprinidae and as such, it is likely that the genes are similar between the two species. Even if the protein is conserved enough to be considered a 5 HT 6 receptor, it is still unknown if its distribution in the brain is similar to that in mammals. If this experiment produces inconclusive results it could mean one of several things three of which are proposed here Either th e receptor is so different that the antagonist does not bind with it (in which case it probably should not be considered a 5 HT 6 receptor), or the receptor is indeed conserved in goldfish, but its distribution in the brain is so different that it provides a different function from that in mammals, or that there is a flaw in the experiment. However,

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26 if this experiment produces positive results, it will provide some of the first substantial evidence that the 5 HT 6 receptor is conserved between humans and gold fish not only in functional structure, but in distribution as well. This could potentially extend the usefulness of the goldfish as a model organism for future research into this receptor as well as filling a gap in the research of serotonin phylogeny Co nsidering the role that the 5 HT 6 receptor has in cognition and its particular interest as a target system for the treatment of neurological disorders, such as AD, this could be quite valuable. There is also a secondary purpose for this experiment. Previou s experiments with SSRIs such as fluoxetine, have suggested that the reduced performance in active learning based tasks may be due, in part, to the anxiolytic effects of serotonin either direct or indirect via allo Some degree of stress is necessa ry for an organism to perform the shuttle box avoidance task Administration of an antagonist to the 5 HT 1a receptor in goldfish has been shown to b e nootropic in the shuttle box paradigm, while administration of fluoxetine impaired their performance (Beulig & Fo wler 2008). A follow up study showed that combined administration of fluoxetine and the 5 HT 1a antagonist drastically impaired the subjects' performance, indicating an unforeseen interaction between the two drugs (Hibberd, 2010) It is possible to infer th at the presumed anxiolytic effects of fluoxetine continue to impair the subjects' performance even when the 5 HT 1a receptor is blocked. This implies that any nootropic properties provided by the antagonist are dependent on normal

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27 function of the other rece ptors, which could mean that 5 HT 1a antagonists partially increase anxiety, thus motivating the subject to actively learn. Alternatively, it could be that blocking the 5 HT 1a receptor is, in fact, a nootropic pathway, independent of the anxiolytic effect of serotonin. However, even with boosted cognition, the anxyolitic effect of serotonin at the other receptors continued to relax the fish to the point that they were unmotivated to perform. This does not explain why performance in this group was lower than with fluoxetine alone. At the risk of anthropomorphizing the fish, with regard to their cognitive processes, a possible analysis is that the anxiolytic effect of fluoxetine, caused them to not care about the shocks, and the nootropic effect of WAY caused them to realize that the shocks were temporary enough that there was no need to avoid them. This scenario is obviously impossible to test without subjective feedback from the subjects. Since goldfish are incapable of such feedback, the only way that this could be tested is in clinical trials with human patients. If a receptor could be isolated which increases cognitive performance independently of stress levels, it would be a valuable target for nootropic drugs. A s mentioned previously, a ntagonists of the 5 HT 6 receptor have been shown to boost performance in memory related tasks in rodents The results of these experiments have implied such antagonists as potential treatment for the (U pton et al 2008). If the performance boost in rodents is simply a result of

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28 increasing stress levels, then this drug would be poorly suited for such a treatment. If, however, blockade of the 5 HT 6 receptor does boost cognition without mitigating the anxiol ytic effect of an SSRI one would expect that a combined administration of fluoxetine with the antagonist would result in both increased cognitive performance and decreased stress. However, the 5 HT 1a receptor is also expressed in the hippocampus, and like 5 HT 6 antagonists, antagonists of 5 HT 1a receptors also increase glutamate and acetylcholine activity in this structure. This would imply that both of these receptors affect memory encoding. By that rationale, a combined treatment of fluoxetine with a 5 H T 6 antagonist, should produce just as much of a decrease in performance as that with 5 HT 1a Perhaps most strangely of all, in studies on rats, co administration of fluoxetine with WAY 100,635 actually improved performance over control in tasks requiring short term memory (Fernandez Perez et al. 2005). In this same experiment, rats administered only WAY showed no significant increase in performance over control. This is quite the opposite of the results o f using these drugs on goldfish, indicating a disti nct difference in the mechanism of serotonin between these taxa. Based on the above research, the hypothesis of this experiment is that goldfish treated with fluoxetine would perform more poorly than control in the shuttle box avoidance task. Furthermore, subjects treated with SB 258,585 would perform better than control in this task. Finally, subjects treated with a co

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29 administration of fluoxetine and SB should perform better than those treated with fluoxetine alone, but worse than those treated with SB al one. Materials and Methods The experimental protocol for this experiment was based heavily on that of previous research conducted on this subject at the Pritzker Marine Biology Research Center espe cially that of last year's senior thesis study (Hibbard, 2 010), the most recent of this line of research Only minor adjustments were made due to budget and time constraints, as well as the experimental variables tested. This section provides a detailed description of the protocol and experimental procedures used for this project. Subjects Forty goldfish (four groups of ten), of the comet variety were purchased from Seascape Aquarium and Pet Center in Sarasota, Florida. The fish ranged from 2 4 inches in length and 6 19 grams in weight, similar in size to those us ed in the previous research. Upon arrival to the Pritzker Lab, the fish were placed in two 55 gallon aquaria for four days in order to monitor their health and make sure they were free of parasites. A single dose of Amquel TM was administered to these tanks to control dangerous nitrogen species. Once it was clear that the fish were all in good health, they were transferred to their home aquaria. Throughout the experiment, a significant number of subjects died for unexplained reasons. Most

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30 of these subjects w ere replaced mid experiment with fish which had been used in previous, similar research (see below in Subject Deaths and Anomalies ) Subjects were randomly assigned to each group. A set of 4 home aquaria was designated to each of the 4 experimental groups: SB (SB 258,585), F (fluoxetine), FSB (combined administration), and CON (control). Subjects were placed into these aquaria prior to their designation into one of these groups. Each group of aquaria was assigned a letter (a d) and each experimental group w as assigned a number (1 4). Letters were matched with numbers via a random number generator, and thus, subjects were assigned to groups. Each group contained 10 subjects, meaning that the first three home aquaria per group each contained three subjects (on e per partition). The fourth aquarium contained only the tenth subject in one of its partitions. After being assigned to their experimental groups, the subjects were numbered, 1 10. For example the 3rd subject in the SB group was designated, SB3, and the 7 th subject in the control group was designated CON7. Home Aquaria and Maintenance For the full duration of the experiment (except during data collection sessions) the fish were housed in 75 liter aquaria divided by opaque Plexiglas into three partitions. I n order to identify individual subjects, the fish were isolated to one per partition. The Plexiglas dividers contained 1 cm holes spaced approximately 5 cm apart. In order to maintain the appropriate drug concentrations in the water (mainly due to budget c onstraints limiting the quantity

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31 of drugs), the aquaria were only filled to a volume of 50 liters which was carefully maintained. The aquaria were filled to the appropriate depth with RO water (reverse osmosis filtered), although the tanks had previously h oused other goldfish, and contained water from them as well. This was done so that the proper nitrogen metabolizing bacteria would already be present in the water when the fish were transferred in. As such, no nitrogen mitigating chemicals were added to th e home aquaria initially On the eighth day of experimental sessions, the tanks were drained to 50% and then refilled with a combination of RO water and tap water. In order to mitigate any nitrogen or chlorine species from the tap water, 1/3 tsp of Amquel TM was added to each tank. Due to a number of subject deaths throughout the first eight days, it was decided on the day following the water change, to add 1 tsp of Erythromycin antibiotic along with 1 tbsp of marine salt to each aquarium. Each partition wa s filtered by a Lustar TM round foam filter. No chemical or charcoal filtration was used due to the concern of it filtering out the drugs. The tanks were aerated via hoses on each filter, connected to the laboratory air pump. Visible detritus was suctioned out via siphon during the mid experiment water change Excessive detritus noticed in any tank throughout the experiment was removed via net. In accordance with the previous experiment, it was decided not to use heating devices to control the temperature of the tanks. The Pritzker Lab is

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32 climate controlled and the water maintains a (nearly) constant 1 9 C (as measured from several home aquaria on multiple days). The tanks were arranged o n a set of shelves against the W est side of the room in three rows. The bottom row was supported off of the floor by foam blocks to prevent any variances in temperature that could be a result of sitting directly on the concrete. Natural sunlight was provided by a large window on the North wall to provide the fish with a normal light/dark cycle. Artificial light from fluorescent bulbs was only used when the experimenter was present. The fish were fed daily by putting a small pinch of TetraMin TM Tropical fish food into each partition. They were then observed to see how much they would eat. If the fish appeared hungry, more food was added. This process was used to avoid overfeeding. Typically, the fish were fed immediately after their sessions in the shuttle boxes. Shuttle Box Aquaria Experimental trials were conducted in shuttle b ox aquaria built by Dr Beulig for all of the previous experiments in this line of research at the Pritzker Lab. There were 8 shuttle boxes available, which allowed for 8 subjects to be tested at a time. The shuttle boxes were constructed of black, opaque, acrylic and measure 11.5 x 40.5 x 16.5 cm. The shuttle box is divided into two equal partitions by a sloped hurdle. During trials, they are filled to approximately 2.5 cm above the hurdle. This is enough so that it is possible for the subjects to cross if motivated

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33 by an appropriate stimulus, but narrow enough that it is unlikely that a subject would cross passively. The bottoms of the shuttle boxes are equipped with drain valves so that the water can easily be replaced between trials. The shuttle boxes wer e housed within wooden cabinets with hinged doors. When closed, the subjects inside are almost completely sealed off from external light sources. The cabinets are equipped with two small, 28V light bulbs, which provide the conditioned stimulus. The shuttle boxes themselves contain conductive wire mesh electrodes on both sides of each partition. These are connected to a Powerstat Variable Autotransformer to provide pulsed, 3 .8 V, AC current to serve as the unconditional stimulus. At the apex of the hurdle, a n infrared photo sensor was positioned to detect if a fish crosses (shuttles) from one side of the shuttle box to the other. The lights, current source, and photo sensors for each shuttle box were connected to a dedicated Colburn Instruments HS Habitest Un iversal Linc box. The Linc boxes were connected to a PC to allow for automated control of the stimuli and data recording.

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34 Fig 5: Schematic diagram of a shuttle box aquarium. From Hibbard, 2010 The PC was equipped with Colburn Instruments Graphic State No tation TM software running on the Microsoft Windows 95 TM operating system. The protocol for presentation of stimuli and data recording was programmed using Graphic State. See Appendix C for the complete programming methodology. The learning performance of each subject was based on the number of avoids, escapes, and failures scored In addition to this, the latency of avoid and escape responses was also measured near the beginning and at the very end of the experiment. This was done to investigate if there w as any change over time in the response time of the subjects to the stimuli. (see Results and Discussion sections). Drug Administration Three of the four experimental groups were administered drugs. One received SB 252,585, one received racemic fluoxetine hydrochloride, and the third received a combined dose of both drugs. The fourth group was a control. As mentioned above, due to the limited budget and high prices of these drugs, the home aquaria were filled to 50 L in order to provide a substantial drug c oncentration using less of each drug in each aquarium. This water volume was maintained across all groups. Both chemicals were obtained from Sigma Aldrich Chemical. A 100 ml solution containing 10 mg of SB 258,585 was prepared, yielding a concentration of 100 g/ml. A plastic pipette was used to transfer 8 ml of this

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35 solution to each 50 L home aquarium in the SB group yielding a concentration of approximately 16 g/L This is consistent with the concentrations used in the previous experiments with other 5 HT receptor antagonists. Since an antagonist inhibits a receptor at a one to one ratio, and this antagonist is highly specific, the dosage should translate between the two. After the mid experiment, 50% water change, 4 ml of this solution was added to each tank to restore the concentration to the same as before the water change. A 100 ml solution containing 100 mg of fluoxetine was prepared, yielding a concentration of 1 mg/ml. Once again, a plastic pipette was used to transfer 8 ml of this solution to each 50 L home aquarium in the fluoxetine group yielding a concentr ation of approximately 160 g/L This is identical to the dose of fluoxetine administered in the previous experiment. After the mid experiment, 50% water change, 4 ml of this solution was adde d to each tank to restore the concentration to the same as before the water change. For the combined group, the same doses described above of each drug were administered to each home aquarium. Concentrations were also replenished after the mid experiment w ater change. For the control group, sham doses of RO water were pipetted into each home aquarium. Drugs were initially administered three days prior to the start of the first experimental session. On the day of the water change, no sessions were run, but t hey were resumed the following day.

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36 Subject Deaths and Anomalies An alarming number of subjects died during the course of the experiment. It is unclear what caused these deaths, but an infection of some sort was suspected, hence their treatment with antibi otics. Stress from the shuttle box sessions may have also played a part. Dead subjects were quickly removed from their aquaria and disposed of using laboratory facilities. So many subjects died that it became necessary to introduce replacement subjects, mi d trial, in order to have any hope of maintaining statistical power Six replacement subjects were added on Session Day 6 and a further eight were added on Session Day 9. Due to time constraints it was not feasible to order more subjects. As such, the repl acement fish were taken from the stock population at the Pritzker Marine Biology Lab. These fish had been used in a previous, unfinished (and cancelled) experiment, and were not naive to the shuttle box. Ironically, they had all undergone exactly 5 session s in the shuttle box, as confirmed by the previous experimenter. As a result, upon their introduction on Session Day 6, the replacement subjects had undergone just as many sessions as all the rest. Admittedly, the replacement subjects introduced on Session Day 9 had undergone 3 fewer sessions than the rest, but since the casualties were distributed nearly evenly across the groups, any affect that this had would be applied across each group. In order to keep the duration of the experiment constrained to the same period of time for all subjects, data from the replacement subjects was combined with data from the corresponding deceased subjects to produce a full set of data.

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37 It should be noted that some of the replacement subjects had been injected with cortisol for the previous experiment. However, this occurred more than 3 months prior to the start of the current experiment, and the previous experimenter could positively assert that all traces of the drug were out the subjects system by this time. It should als o be noted that most of the replacement subjects were slightly larger than their initial subject counterparts (3 4 inches rather than 2 3 for the initial subjects ), but were still all of the comet variety. Subject deaths occurred on nearly every day of the experiment. As such, even after replacement subjects were introduced, there were gaps in the data for days between subject death and replacement. To compensate for this, two make up sessions were conducted on the two days after the 16 main sessions. On th ese days, only subjects with gaps in their data were run. As a result of this, not all sessions of the same number were conducted on the same day across all subjects There were some cases in which a subject was missing more than 2 sessions worth of data. Since the statistical analysis could not be performed if there were gaps in the data, these remaining gaps were filled with "dummy data." Dummy data was calculated by finding the mean score for the given block from the subjects in that group with complete sets of data (including those with make up sessions), then dividing this mean by 4 to give a single day's score (rounded to the nearest whole number). Dummy data were calculated for (and used in)

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38 each of the 4 blocks within each of the 4 subject groups (s ee Appendix A for complete data for all sessions for each subject) Apart from subject mortality, there were also several gaps produced by equipment malfunctions. There were two shuttle boxes with malfunctioning photo sensors. These boxes were not used aft er the problem was discovered and were quickly repaired for future sessions, but not before a few subjects were run in them. In another, isolated case, the shock delivery system became detached from one of the shuttle boxes. Data for subjects run on malfun ctioning equipment was discarded and make up sessions (or dummy data if necessary) was used to provide these subjects with complete sets of data. The only caveat to this situation is that, since the subjects were actually in the shuttle boxes during the di scarded sessions, once the make up sessions were conducted, these subjects were in the shuttle boxes more times than the rest (up to 18 sessions vs. 16). Finally, there is the case of subject FSB5 who, on 5 separate occasions, jumped out of the shuttle box mid session. Again, make up sessions and dummy data were used to compensate. Despite all of these issues, overall impact on the results should be minimal due to the fairly even distribution of these problems over all 4 subject groups. For a complete accou nt of all subject deaths, equipment malfunctions and anomalies see Appendix B.

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39 Experimental Protocol Protocol for each experimental session day consisted of checking for dead subjects, testing the equipment, and actually running the sessions with subjects There were 16 days of experimental sessions over a period of 17 days (no data was collected on the day of the water change), in which each subject was run once. Within each session, each subject was run through 15 trials. Most sessions were started at ap proximately 1:00 PM, although due to some scheduling conflicts, a few sessions were started later at night. Once started, a complete session required about 3.5 hours. Since there were 8 shuttle boxes, each session consisted of 5 runs, with 8 fish each so that all 40 fish were run in each daily session. At the beginning of a daily session, before any subjects were run, each shuttle box was tested using a specific program in which the shock and lights could be turned on and off by tripping the photo sensor by hand. This allowed the photo sensor, lights, and with the use of a voltmeter, the shock delivery system to be tested before any subjects were placed in the shuttle box that day. Before any experimental sessions were conducted, 2 large drums were filled halfway with tap water. These were connected via a PVC siphon. Another PVC siphon, ending in a valve, was placed in the second of these drums and served as a tap with which to draw water from. These drums served as the reservoirs for water used in the shu ttle boxes during experimental sessions. The first drum was equipped with a large, charcoal filter. After use in the shuttle

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40 boxes, the water would be poured back into this drum, so as to filter out any stress or fear pheromones which could be detected by the fish, and might interfere with the experiment. Each run of 8 subjects began with the filling of the shuttle boxes with water from the second (cleaner) drum. Then, the program was set up in Graphic State. Next, the appropriate subjects were carefully re moved from their home aquaria using a hand net, and placed in their respective shuttle boxes. The doors to the shuttle boxes were then closed so that the only light available to the fish would come from the conditional stimulus. Once all subjects were in p lace and the doors closed, the program would be started. As soon as the program finished with each individual subject, it would be removed by net from the shuttle box and returned to its home aquarium. Then the shuttle boxes were drained and the water, pou red into the first drum for filtration. The entire process was then repeated to reset the equipment for the next round of subjects. Once all 8 subjects had been returned to their respective home aquaria, they were fed. Each session for on e subject took bet ween 10 and 29 minutes, depending on the duration of the randomized inter trial interval (S1). Results Avoidance Responses All 10 subjects in each of the four groups underwent 16 sessions. These sessions were further grouped into 4 "blocks" of 4 days, each

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41 As with the previous experiments, there was a high degree of variability within each group. However, the general trend can be summed up thus: the fluoxetine group performed the most poorly, the SB and fluoxetine combined group performed slightly better, the SB group performed slightly better than that, and control group outperformed all three by a fairly wide margin (see Figure 6). F ig 6: Mean avoids per session for each group, plotted over all sessions. Table 1 contains the total responses over all se ssions for each individual subject. This illustrates the high degree of variability between subjects within each group. Table 1: Total avoids for each subject summed over all sessions and mean score for each group. Total Avoids Over All Sessions Subject A voids Subject Avoids Subject Avoids Subject Avoids SB1 10 FSB1 32 F1 7 CON1 124 0 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Mean # of Avoids Session # Mean Avoids Over All Sessions SB FSB F CON

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42 SB2 126 FSB2 60 F2 25 CON2 112 SB3 69 FSB3 99 F3 72 CON3 16 SB4 101 FSB4 39 F4 20 CON4 138 SB5 50 FSB5 75 F5 0 CON5 115 SB6 88 FSB6 17 F6 13 CON6 82 SB7 23 FSB7 79 F7 51 CON7 99 SB8 48 FSB8 9 F8 77 CON8 35 SB9 100 FSB9 130 F9 131 CON9 69 SB10 84 FSB10 49 F10 106 CON10 121 Mean 69.9 Mean 58.9 Mean 50.2 Mean 91.1 Table 2 provides descriptive statistics on the total avoids of the subjects in each of the 4 groups over a ll sessions. Table 2: Descriptive statistics of total avoids over all sessions for each group. SB SB and Fluoxetine Fluoxetine Control Mean 69.90 58.90 50.20 91.10 Standard Deviation 36.83 37.70 44.91 40.28 Min 10 9 0 16 Max 126 130 131 138 N 10 10 1 0 10 The control group had the highest mean number of avoids (91.10) as well as the subject that performed the greatest number of total avoids over all sessions (138) The fluoxetine group had the lowest mean number of avoids (50.20) and the subject that performed the fewest total avoids over all sessions

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43 (0) This was the only subject out of all 4 groups which failed to perform a single avoid. As previously stated, variance within groups was fairly high. For the fluoxetine group, the standard deviation w as nearly 90% the value of the mean. Variance between subjects within the SB and control groups was somewhat lower, with the standard deviation roughly half the value of the mean. A two way ANOVA with repeated measures was performed for avoid responses of groups over blocks, using the SAS v9.2 software. In addition, Tukey's HSD post hoc test was performed to further analyze the data. Significance was found for both blocks and groups. The F value for blocks was 14.89 and the p value was found to be <0.0001. The F value for groups was 5.67 and the p value was 0.0011. The mean avoid responses between groups with significant differences at the 0.05 level over blocks of time can be found in Table 3. Although no significance was found between any of the treated gr oups, both the fluoxetine group and the SB and fluoxetine combined group were found to be significantly different from the control group. Furthermore, although no significance was found between Blocks 2 4, Block 1 was significantly different from the other three. Table 3: Mean avoids per subject, per block with Tukey's HSD data. Means with the same letters have significant difference at the 0.05 value. Mean Avoids SB SB and Fluoxetine Fluoxetine Control Block 1 7.3 A, E,I 8.4 B, F,J,M 2.6 C, G,K,N 7.9 D H,L,M,N Block 2 19.5 A 12.3 B O 14.9 C P 23.3 D, O,P

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44 Block 3 20.7 E, 19.7 F,Q 16.7 G,R 27.2 H,Q,R Block 4 22.4 I, 18.5 J,S 16 K,T 32.5 L,S,T All Blocks 69.9 U 58.9 V 50.2 W 91.1 V,W F igure 7 compares the mean avoid responses of each group across blocks. Fig 7: Mean avoids per subject for each group across all blocks. Failure Responses Failures were analyzed in the same manner as avoids. Failure results showed an even higher degree of variability than the avoid results. As such, generalizations are somewhat difficult to mak e, although the fluoxetine group had the highest rate of failures, while the control group had the lowest. The control group is noteworthy in that the degree of variability was lower than the other 0 5 10 15 20 25 30 35 1 2 3 4 Mean # of Avoids Block Mean Avoids Over Blocks SB FSB F CON

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45 groups and the failure rate was very low throughout the en tire experiment. For all three treated groups, a trend can be seen of increasing failure rate as the experiment progressed (see Figure 8). Fig 8 : Mean failures per session for each group, plotted over all sessions. Table 3 contains the total responses ov er all sessions for each individual subject. Again variability between subjects, within groups was very high. Again the control group is noteworthy, as it is not only the group with the lowest mean failures (by a considerable margin), but it is also the on ly group with subjects who never performed a single failure. Table 4: Total failures for each subject summed over all sessions and mean score for each group. Total Failures Over All Sessions Subject Failures Subject Failures Subject Failures Subject Failu res SB1 82 FSB1 104 F1 20 CON1 1 SB2 1 FSB2 26 F2 68 CON2 0 0 0.5 1 1.5 2 2.5 3 3.5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Mean # of Failures Session # Mean Failures Over All Sessions SB FSB F CON

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46 SB3 3 FSB3 21 F3 3 CON3 8 SB4 8 FSB4 4 F4 56 CON4 0 SB5 6 FSB5 10 F5 113 CON5 0 SB6 8 FSB6 20 F6 102 CON6 0 SB7 16 FSB7 11 F7 11 CON7 3 SB8 51 FSB8 113 F8 9 CON8 6 SB9 3 FSB9 2 F9 1 CON9 1 SB10 11 FSB10 3 F10 8 CON10 4 Mean 18.9 Mean 31.4 Mean 39.1 Mean 2.3 Table 5 provides descriptive statistics on the total failures of the subjects in each of the 4 groups over all sessions. Table 5: Descriptive statistics of total failures over all s essions for each group. It is clear how high the degree of variance between subjects was for failur es, as the standard deviation is greater than the mean in all four groups. The fluoxetine and the SB and fluoxetine combined group were tied for the subject with the most failures (113), while the control group not only had the SB SB and Fluoxetine Fluoxetine Control Mean 18.9 31.4 39.1 2.3 Standard Deviation 26.48 41.49 42.58 2.87 Min 1 2 1 0 Max 82 113 113 8 N 10 10 10 10

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47 subject with the fewest fail ures, but several subjects with 0 total failures for the entire experiment. Like the avoidance responses, a two way ANOVA was performed for failure responses of groups over blocks. Tukey's HSD post hoc test was also performed to further analyze the data. N o significance was found between blocks, but significance was found between groups. The F value for blocks was 0.92 with a p value of 0.4322. The F value for groups was 6.29 and the p value was 0.0005. The mean failure responses between groups with signifi cant differences at the 0.05 level over blocks of time can be found in Table 6. As with avoidance responses, no significance was found between any of the treated groups, but both the fluoxetine group and the SB and fluoxetine combined group were found to b e significantly different from the control group. Table 6: Mean failures per subject, per block with Tukey's HSD data. Means with the same letters have significant difference at the 0.05 value. Mean Failures SB SB and Fluoxetine Fluoxetine Control Block 1 1.7 A 5.2 B 6.7 C 0.5 B,C Block 2 4.6 D 9.8 E 9.3 F 0.8 E,F Block 3 5.8 G 10.1 H 11.8 I 0.4 H,I Block 4 6.8 J 6.3 K 11 L 0.4 K,L All Blocks 18.9 M 31.4 N 39.1 O 2.3 N,O

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48 Figure 9 compares the mean failure responses of each group across blocks. Fig 9: Mean failures per subject fo r each group across all blocks. Escape Responses Escape responses are less important to the analysis of results, as any response that is not an avoid or failure must be an escape. However, the total escapes for each subject are provided in Tabl e 7, while descriptive statistics of escape responses can be found in Table 8. Table 7: Total escapes for each subject summed over all sessions and mean score for each group. Total Escapes Over All Sessions Subject Escape s Subject Escape s Subject Escape s Subject Escape s SB1 148 FSB1 104 F1 213 CON1 115 0 2 4 6 8 10 12 14 1 2 3 4 Mean # of Failures Blocks Mean Failures Over Blocks SB FSB F CON

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49 SB2 113 FSB2 154 F2 147 CON2 128 SB3 168 FSB3 120 F3 165 CON3 216 SB4 131 FSB4 197 F4 164 CON4 102 SB5 184 FSB5 155 F5 127 CON5 125 SB6 144 FSB6 203 F6 125 CON6 158 SB7 201 FSB7 150 F7 178 CON7 138 S B8 141 FSB8 118 F8 154 CON8 199 SB9 137 FSB9 108 F9 108 CON9 170 SB10 145 FSB10 188 F10 126 CON10 115 Mean 151.2 Mean 149.7 Mean 150.7 Mean 146.6 Table 8: Descriptive statistics of total escapes over all sessions for each group. SB SB and Fluoxetine Fluoxetine Control Mean 151.2 149.7 150.7 146.6 Standard Deviation 26.06 36.98 30.75 38.17 Min 113 104 108 102 Max 201 203 213 216 N 10 10 10 10 The mean number of total escapes were nearly identical between all four groups, although technically the SB group had the highest and the control group had the lowest. The control group had the subject with the most total escapes and the subject with the fewest total escapes. Avoid Latency Although the primary measure of learning ability in this experiment w as the o verall number of avoids, the latency of the response for each trial was also

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50 measured. Two sessions were sampled for analysis: Session 5, at the beginning of Block 2, and Session 16, at the end of the entire experiment. This was done to investigate if there was any change over time in the response time of the subjects for each gro up to the conditional stimulus. Figure 10 illustrates the distribution of the mean response time for each group at Session 5, while Figure 11 illustrates the same for Sessi on 16. Fig 10: Distribution across duration of the conditional stimulus of avoid latency for all groups at Session 5. 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1 2 3 4 5 6 7 8 9 10 Mean # of Avoids Seconds Distribution of Avoid Latency at Session 5 SB FSB F CON

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51 Fig 11: Distribution across duration of the conditional stimulus of avoid latency for all groups at Session 16. Comparison of the two sessions reveals a noticeable shift toward late avoid responses at Session 16, as opposed to mid duration ones at Session 5. This is especially evident in the control group, which at Session 5, scored most of its avoids 3 seconds into the conditional stim ulus, and at Session 16, scored most of its avoids between 6 and 10 seconds in. Besides this, all of the groups scored the fewest avoids early in the duration of the stimulus at both Sessions except for a slight trend towards second 3 in the fluoxetine gr oup. Because there were different numbers of total avoids in each group between the two sessions, a statistical analysis of the latency data would have been impractical, and was not attempted. 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 1 2 3 4 5 6 7 8 9 10 Mean # of Avoids Seconds Distribution of Avoid Latency at Session 16 SB FSB F CON

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52 Escape Latency Escape latency was also analyzed in a similar ma nner to avoid latency, to investigate if there was any change over time in the response time of the subjects for each group to the unconditional stimulus. Again, only Sessions 5 and 16 were sampled. Figure 12 illustrates the distribution of the mean respon se time for each group at Session 5, while Figure 13 illustrates the same for Session 16. Fig 12: Distribution across duration of the un conditional stimulus of escape latency for all groups at Session 5. 0 1 2 3 4 5 6 1 2 3 4 5 Mean # of Escapes Seconds Distribution of Escape Latency at Session 5 SB FSB F CON

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53 Fig 13: Distribution across duration of the unc onditional stimulus of escape latency for all groups at Session 16. As shown in the graphs, above, there is virtually no difference escape latency between the two sessions. For both sessions, escapes are exponentially higher at the beginning of the stimulu s than at the end. Again, since there were different numbers of total escapes between the two sessions, no statistical tests were performed. Discussion The results of this experiment were rather surprising. All three treated groups performed fewer avoids a nd more failures than the control group, indicating that memory and learning ability was impaired by both drugs (individually and together) and not just fluoxetine This is inconsistent with previous research on 5 HT 6 receptor antagonists in rats (Dawson et al. 2001) 0 1 2 3 4 5 6 1 2 3 4 5 Mean # of Escapes Seconds Distribution of Escape Latency at Session 16 SB FSB F CON

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54 Also surprising was the fact that while the group treated with SB 258,585 performed worse than the control, the SB and fluoxetine combined treatment group performed slightly better than the group treated with just fluoxetine. However, the dif ference between the latter two groups was not found to be statistically significant. Furthermore, although the impairment of performance by the SB treated group is interesting, it was not found to be statistically significant. Avoidance learning in this pa radigm is well supported by the results. Although there was a gradual increase in the number of avoids over all four blocks of time, the only significant difference was between Block 1 and the other three blocks. This indicates that learni ng happens most r apidly within the first several sessions. This contrasts with the previous study (Hibberd, 2010), which showed a much more gradual learning curve. With regard to failures, no significant difference could be found in failure frequency over time. This is due in large part to the high variance between subjects within groups. The comparison between groups was similar for failures as for avoids. The control group performed very few total failures throughout the entire experiment. Its performance was found to be significantly different from the fluoxetine treated group and the combined treatment group. The SB treated group performed the best among the drug treated groups (for avoids as well as failures) but not as well as the control. In fact the SB group had such a median level of performance that there was no significance found between it and any of the other groups (for avoids or failures).

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55 Performance Variability Within each group, there was a great deal of variance in performance between subjects. Each group h ad at least one or two subjects that performed extremely well (100 or more total avoids and virtually no failures), and one or two subjects that performed very poorly (almost no avoids and many failures). There are several factors worth considering that m ay have contributed to this high variance, most of which can probably be ruled out. The most obvious explanation is that there was some unforeseen environmental factor affecting the subjects. However, all subjects were housed in identical aquaria, in the s ame room, at the same temperature, and fed the same amount. All subjects, including those which replaced the ones that died, were originally obtained from the same source. One major difference was that the replacement subjects had been housed in a goldfish pond under the Pritzker Marine Biology Lab for approximately 3 months prior to their introduction to this experiment. As mentioned previously, at the time of the first introduction of replacements, the new subjects had undergone exactly the same number of sessions in the shuttle box (5) from a previous experiment, as the rest of the subjects in this experiment. The same cannot be said for those subjects in the second round of replacements, as these had still only undergone 5 sessions, while the rest had, at this point, undergone 8 sessions. It is also worth noting that all of the replacement subjects had been injected with cortisol for the previous experiment (approximately 3 months prior), although as mentioned earlier, the previous experimenter was able to positively assert that all traces of cortisol were

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56 out of these subjects' systems by the time they were introduced as replacements. Another drawback of introducing replacements mid trial is that the new subjects had been exposed to the drugs for a short er period of time than the initial subjects. Despite all of these possible contributing factors, there was no observable difference between the overall performance of the initial subjects and the replacement subjects. Some of the replacements performed ver y well, while others performed very poorly. Also, the subjects which died were distributed fairly evenly across all groups. This not only indicates that no specific treatment contributed to their deaths, but also that any effect on the data by replacing su bjects did not impact any one group more than the others. Another factor to consider would be the testing equipment. Although the shuttle boxes were drained and refilled between each subject, the total amount of water recycled for use in the shuttle boxes was not enough complete an entire day's worth of sessions, and the filtration of this water was not fast enough to keep up with the rate of replacement. This means that as the sessions progressed each day, the replacement water was slowly mixed with the pa rtially filtered, used water. However, any effect this had must have been small, as there was no observed difference between the performance of subjects run in the early sessions of the day and those run later. The lights, shock delivery, and photo sensor s of each shuttle box were tested every day before sessions. This helped minimize equipment malfunctions,

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57 but was far from perfect. There were several occasions in which some aspect of the equipment stopped working mid way through the sessions of the day. For a complete list of such malfunctions, see Appendix B. Since such malfunctions prevented data collection from those subjects in the shuttle box, that day, two make up days were added after the first 16 so that those subjects missing data would have a co mplete set of 16 sessions. This leads to the next possible contributing factor. Because sessions were missed (due to days between subject death and replacement or equipment malfunction), and subsequently made up, one subject's Session 5, for example, may h ave occurred on a different day than another subject's Session 5. A strong effort was made to maintain the same environment and routine day to day. The only day s in which there was any deviation from the protocol, w ere the water change day, between Session Days 7 and 8, in which no sessions were conducted and the following day in which the subjects were treated with antibiotics The water change day was also the day that the tanks were treated with Amquel TM Also, in order to maintain the same drug concent ration in the treated aquaria, drugs had to be re administered. It is possible that there was a subtle variation in the concentration of the drugs before and after the water change. Since no specific days were noticed to have any impact on overall performa nce, and no change in overall performance was noted after the water change or antibiotic treatment, any impact on the results, due to th e shift in session days is probably minimal.

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58 There were also some subjects which had more than two sessions worth of mi ssing data. As mentioned previously, any additional gaps in data were filled in with dummy data. Since the number of these such sessions was small, and the dummy data itself was based on the remaining subjects' mean data for the given block, the effect was probably small, but almost certainly still contributed to the variance to some degree. Of much greater influence, may have been the overall health of the subjects during the experiment. Since so many subjects died over the course of the experiment, it wa s assumed that they were infected with something. Many of the subjects that scored a lot of escapes but few avoids, may have been too sick to effectively learn the avoidance task, but still capable of moving when shocked. The final possible contributing fa ctor to the variability between subjects is probably the most likely to have had the greatest impact, and was also the most unavoidable. Although the subjects were all goldfish of the comet variety, obtained from the same supplier, there appears to be a sh arp difference in the base cognitive abilities of each subject, regardless of environment or drug treatment. The fact of the matter seems to be that some goldfish are simply better at the shuttle box avoidance task than others. The only way to compensate f or this fact would be to greatly increase the number of subjects used in each group, which was not feasible for this experiment due to budget and time constraints.

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59 Support of Hypotheses and Conclusions At the outset of this study, the experimenter was atte mpting to investigate two major things. The first was evidence that Carassius auratus possessed a 5 HT 6 receptor. The second was, if so, are the nootropic effects of blocking this receptor pathway affected by stress levels (as tested b y administration of f luoxetine)? To the first question, there is some evidence provided that goldfish do, in fact, possess the 5 HT 6 receptor in some capacity. Although there was no statistical significance between the performance of the control group and the SB treated group, there was a consistent general trend of poorer performance in the SB treated group. There are several possible explanations offered for this result. The first is that the 5 HT 6 receptor has a different distribution in the brain of goldfish than it does i n roden ts. A radioligand binding assay, or immuno localization assay would be necessary to map the full distribution of this receptor in the goldfish brain. A second explanation is that the distribution of the 5 HT 6 receptor is similar in goldfish as it is in mammals, but the difference lies in the amino acid sequence the receptor itself. As previously mentioned, the zebrafish genome was found to contain a gene for a protein with a 54% similarity to the human 5 HT 6 receptor (Best & Alderton 2008). Since a c hange of one amino acid can potentially render a protein non functional, it is not unreasonable to consider that a change of this magnitude could alter the binding properties of the receptor. It is possible that, with the goldfish 5 HT 6 receptor, SB 258,58 5 acts, not as an

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60 antagonist, but an agonist. This would amplify the receptor pathway rather than block it, and thus, cause a reduction in memory rather than an increase. Confirmation of this would require isolation of the goldfish 5 HT 6 receptor and a sub sequent receptor binding assay. It is also possible that there is something else at work that is, as of yet, not fully understood. The serotonin system is one of the most complex systems in the brain. Although most research has implied 5 HT 6 antagonists as nootropic, one even reported an anxiolytic or antidepressant effect (Wesoloska et al. 2007) which would place it in the same profile as fluoxetine. Clearly, a great deal of research is still required in order to understand this system. Perhaps one of the newer 5 HT 6 receptor antagonists, such as SB 399,885 (Hirst et al. 2006) would provide more information. Although there was no significance between the SB group and any of the treated groups, the fluoxetine group still performed the worst, while the SB and fluoxetine combined treatment group performed somewhere between the two. This is also a strange outcome, but without statistical significance, it is difficult to comment on it further. Finally, it is worth mentioning, that this experiment is the first in this line of research on goldfish, that has shown a statistically significant difference between the fluoxetine treated group and the control group. It is now more clear than ever, that the anxiolytic effect of this drug impair s learning and memory perform ance, reinforcing the assertion that some stress is necessary in order to motivate an

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61 organism to learn. Further research is still needed to ascertain if fluoxetine exerts its effects through the serotonin system or through the recently proposed Allo path way (Pinna et al. 2009). Although the results were somewhat different than those predicted by the hypothesis, overall this study was a success. Direct investigations of the pharmacodynamics of the 5 HT 6 receptor would be advisable to expand on this study, as well as investigations of the serotonergic systems of other species such as those among chondrichthyes, amphibians, or even reptiles, in order to expand the knowledge of serotonin phylogeny Finally a firm understanding of the mechanism of fluoxetine wo uld be extremely valuable to this line of research. All of these proposed studies hold great potential.

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62 Appendix A: Raw Data Avoidance, Escape, and Failure Responses The following lists all raw data for all subjects in order of session. Data marked with an asterisk indicates dummy data, used to fill gaps with missing data due to subject death, equipment malfunction, etc. Session 1 Subject Avoids Escapes Failures Subject Avoids Escapes Failures SB1 0 15 0 FSB1 0 15 0 SB2 0 15 0 FSB2 0 15 0 SB3 0 14 1 FSB3 1 14 0 SB4 0 15 0 FSB4 0 15 0 SB5 0 15 0 FSB5 0 15 0 SB6 0 15 0 FSB6 0 11 4 SB7 0 15 0 FSB7 0 13 2 SB8 0 15 0 FSB8 0 15 0 SB9 0 15 0 FSB9 0 14 1 SB10 0 15 0 FSB10 0 13 2 TOTAL 0 149 1 TOTAL 1 140 9 Subject Avoids Escapes Failures Subject Avoids Escapes Failures F1 0 13 2 CON1 0 15 0 F2 0 13 2 CON2 0 15 0 F3 0 14 1 CON3 2 13 0 F4 0 8 7 CON4 0 15 0 F5 0 14 1 CON5 0 15 0 F6 0 15 0 CON6 0 15 0 F7 0 15 0 CON7 0 14 1 F8 0 13 2 CON8 0 15 0 F9 0 15 0 CON9 0 15 0 F10 0 12 3 CON10 0 14 1 TOTAL 0 132 18 TOTAL 0 146 2 Session 2

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63 Subject Avoids Escapes Failures Subject Avoids Escapes Failures SB1 0 15 0 FSB1 5 9 1 SB2 3 11 1 FSB2 0 13 2 SB3 0 15 0 FSB3 7 7 1 SB4 1 7 7 FSB4 0 15 0 SB5 2 13 0 FSB5 2 13 0 SB6 2 12 1 FSB6 0 10 5 SB7 0 15 0 FSB7 0 15 0 SB8 1 13 1 FSB8 0 14 1 SB9 0 15 0 FSB9 3 12 0 SB10 0 14 1 FSB10 0 15 0 TOTAL 9 130 11 TOTAL 17 123 10 Subject Avoids Escapes Failures Subject Avoids Escapes Failures F1 0 13 2 CON1 2 13 0 F2 0 12 3 CO N2 2 13 0 F3 4 11 0 CON3 2 13 0 F4 0 8 7 CON4 5 10 0 F5 0 14 1 CON5 1 14 0 F6 0 15 0 CON6 1 14 0 F7 1 14 0 CON7 1 14 0 F8 0 15 0 CON8 0 15 0 F9 0 15 0 CON9 0 15 0 F10 0 15 0 CON10 1 14 0 TOTAL 5 132 13 TOTAL 15 135 0 Session 3 Subject A voids Escapes Failures Subject Avoids Escapes Failures SB1 0 15 0 FSB1 3 12 0 SB2 10 5 0 FSB2 1 10 4 SB3 2 12 1 FSB3 13 1 1 SB4 2 13 0 FSB4 1 14 0 SB5 2 13 0 FSB5 3 12 0 SB6 0 15 0 FSB6 0 11 4 SB7 0 15 0 FSB7 4 10 1 SB8 2 13 0 FSB8 1 12 2 SB9 2 13 0 FSB9 5 10 0

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64 SB10 3 12 0 FSB10 0 15 0 TOTAL 23 126 1 TOTAL 31 107 12 Subject Avoids Escapes Failures Subject Avoids Escapes Failures F1 0 15 0 CON1 5 10 0 F2 0 13 2 CON2 5 10 0 F3 3 12 0 CON3 2 13 0 F4 0 3 12 CON4 2 13 0 F5 0 14 1 CON5 2 13 0 F6 1 14 0 CON6 2 13 0 F7 0 15 0 CON7 1 13 1 F8 0 13 2 CON8 1 14 0 F9 5 10 0 CON9 0 15 0 F10 0 13 2 CON10 6 8 1 TOTAL 9 122 19 TOTAL 26 122 2 Session 4 Subject Avoids Escapes Failures Subject Avoids Escapes Failures SB1 2 12 1 FSB1 0 11 4 SB2 8 7 0 FSB2 1 10 4 SB3 3 11 1 FSB3 11 4 0 SB4 6 9 0 FSB4 2 13 0 SB5 4 11 0 FSB5 4 10 1 SB6 5 10 0 FSB6 0 13 2 SB7 0 15 0 FSB7 5 10 0 SB8 3 11 1 FSB8 0 6 9 SB9 4 10 1 FSB9 9 6 0 SB10 6 9 0 FSB10 3 11 1 TOTAL 41 105 4 TO TAL 35 94 21 Subject Avoids Escapes Failures Subject Avoids Escapes Failures F1 0 15 0 CON1 2 13 0 F2 0 11 4 CON2 2 13 0 F3 3 11 1 CON3 0 15 0 F4 0 12 3 CON4 4 11 0 F5 0 13 2 CON5 4 11 0

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65 F6 0 15 0 CON6 9 6 0 F7 2 13 0 CON7 6 9 0 F8 0 11 4 CON8 3 12 0 F9 7 8 0 CON9 0 14 1 F10 0 12 3 CON10 8 7 0 TOTAL 12 121 17 TOTAL 38 111 1 Session 5 Subject Avoids Escapes Failures Subject Avoids Escapes Failures SB1 1 14 0 FSB1 1 8 6 SB2 10 5 0 FSB2 0 14 1 SB3 4 11 0 FSB3 7 7 1 SB4 7 8 0 FSB4 2 12 1 SB5 2 11 2 FSB5 6 9 0 SB6 7 8 0 FSB6 0 15 0 SB7 0 13 2 FSB7 2 13 0 SB8 3 12 0 FSB8 0 6 9 SB9 9 6 0 FSB9 9 5 1 SB10 2 13 0 FSB10 1 14 0 TOTAL 45 101 4 TOTAL 28 103 19 Subject Avoids Escapes Failures Subject Avoids Escapes Failu res F1 0 15 0 CON1 10 5 0 F2 0 10 5 CON2 10 5 0 F3 3 12 0 CON3 1 12 2 F4 7 7 1 CON4 5 10 0 F5 0 13 2 CON5 4 11 0 F6 0 3 12 CON6 9 6 0 F7 6 9 0 CON7 6 9 0 F8 0 15 0 CON8 0 14 1 F9 10 5 0 CON9 1 14 0 F10 1 14 0 CON10 5 8 2 TOTAL 27 103 2 0 TOTAL 51 94 5 Session 6

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66 Subject Avoids Escapes Failures Subject Avoids Escapes Failures SB1 4 11 0 FSB1 1 4 10 SB2 7 8 0 FSB2 0 13 2 SB3 5 10 0 FSB3 5 10 0 SB4 8 7 0 FSB4 3 12 0 SB5 2 12 1 FSB5 4 11 0 SB6 7 8 0 FSB6 0 12 3 SB7 5 9 1 FSB 7 3 10 2 SB8 6 8 1 FSB8 0 4 11 SB9 6 8 1 FSB9 6 9 0 SB10 2 11 2 FSB10 5 10 0 TOTAL 52 92 6 TOTAL 27 95 28 Subject Avoids Escapes Failures Subject Avoids Escapes Failures F1 1 12 2 CON1 10 5 0 F2 4 9 2 CON2 10 5 0 F3 4 10 1 CON3 1 13 1 F4 5 7 3 CON4 7 8 0 F5 0 14 1 CON5 8 7 0 F6 0 3 12 CON6 3 12 0 F7 3 12 0 CON7 5 10 0 F8 11 4 0 CON8 3 11 1 F9 9 6 0 CON9 0 15 0 F10 8 7 0 CON10 7 8 0 TOTAL 45 84 21 TOTAL 54 94 2 Session 7 Subject Avoids Escapes Failures Subject Avoids Escap es Failures SB1 2 7 6 FSB1 0 5 10 SB2 8 7 0 FSB2 3 11 1 SB3 7 8 0 FSB3 4 10 1 SB4 9 6 0 FSB4 2 12 1 SB5 2 13 0 FSB5 3 10 2 SB6 4 11 0 FSB6 1 14 0 SB7 2 7 6 FSB7 1 12 2 SB8 1 9 5 FSB8 2 5 8 SB9 6 9 0 FSB9 9 6 0

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67 SB10 6 9 0 FSB10 3 12 0 T OTAL 47 86 17 TOTAL 28 97 25 Subject Avoids Escapes Failures Subject Avoids Escapes Failures F1 0 15 0 CON1 11 4 0 F2 0 8 7 CON2 9 6 0 F3 5 10 0 CON3 2 12 1 F4 4 8 3 CON4 14 1 0 F5 0 14 1 CON5 11 4 0 F6 1 6 8 CON6 1 14 0 F7 3 12 0 CON7 7 8 0 F8 8 7 0 CON8 4 11 0 F9 10 5 0 CON9 2 13 0 F10 8 7 0 CON10 1 14 0 TOTAL 39 92 19 TOTAL 62 87 1 Session 8 Subject Avoids Escapes Failures Subject Avoids Escapes Failures SB1 0 7 8 FSB1 0 3 12 SB2 8 7 0 FSB2 4 7 4 SB3 7 8 0 FSB3 10 4 1 SB4 8 7 0 FSB4 2 13 0 SB5 3 12 0 FSB5 3 10 2 SB6 4 10 1 FSB6 1 14 0 SB7 1 12 2 FSB7 7 8 0 SB8 2 5 8 FSB8 1 7 7 SB9 8 7 0 FSB9 9 6 0 SB10 10 5 0 FSB10 3 12 0 TOTAL 51 80 19 TOTAL 40 84 26 Subject Avoids Escapes Failures Subject Avoids Esca pes Failures F1 2 12 1 CON1 7 8 0 F2 2 6 7 CON2 12 3 0 F3 4 11 0 CON3 1 14 0 F4 0 15 0 CON4 12 3 0 F5 0 4 11 CON5 11 4 0

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68 F6 0 2 13 CON6 1 14 0 F7 2 13 0 CON7 11 4 0 F8 7 7 1 CON8 5 10 0 F9 9 6 0 CON9 1 14 0 F10 12 3 0 CON10 5 10 0 TOTA L 38 79 33 TOTAL 66 84 0 Session 9 Subject Avoids Escapes Failures Subject Avoids Escapes Failures SB1 0 5 10 FSB1 4 2 9 SB2 6 9 0 FSB2 3 11 1 SB3 5 10 0 FSB3 10 5 0 SB4 4 11 0 FSB4 5 10 0 SB5 5 9 1 FSB5 9 4 2 SB6 5 10 0 FSB6 1 14 0 SB7 0 14 1 FSB7 7 5 3 SB8 5 5 5 FSB8 0 5 10 SB9 6 9 0 FSB9 10 5 0 SB10 10 5 0 FSB10 4 11 0 TOTAL 46 87 17 TOTAL 53 72 25 Subject Avoids Escapes Failures Subject Avoids Escapes Failures F1 0 12 3 CON1 11 4 0 F2 2 7 6 CON2 6 9 0 F3 5 10 0 CON3 2 13 0 F4 2 13 0 CON4 9 6 0 F5 0 2 13 CON5 7 8 0 F6 1 7 7 CON6 6 9 0 F7 3 12 0 CON7 7 8 0 F8 7 8 0 CON8 0 15 0 F9 9 5 1 CON9 1 14 0 F10 12 3 0 CON10 11 4 0 TOTAL 41 79 30 TOTAL 60 90 0 Session 10

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69 Subject Avoids Escapes Failures Subject Avo ids Escapes Failures SB1 0 8 7 FSB1 5 4 6 SB2 9 6 0 FSB2 4 9 2 SB3 2 13 0 FSB3 1 6 8 SB4 9 6 0 FSB4 6 9 0 SB5 3 12 0 FSB5 4 11 0 SB6 9 6 0 FSB6 3 12 0 SB7 1 13 1 FSB7 8 7 0 SB8 6 6 3 FSB8 0 3 12 SB9 6 9 0 FSB9 9 6 0 SB10 6 9 0 FSB10 5 1 0 0 TOTAL 51 88 11 TOTAL 45 77 28 Subject Avoids Escapes Failures Subject Avoids Escapes Failures F1 1 14 0 CON1 8 6 1 F2 2 4 9 CON2 8 7 0 F3 4 11 0 CON3 0 13 2 F4 0 12 0 CON4 11 4 0 F5 0 5 10 CON5 10 5 0 F6 1 7 7 CON6 6 9 0 F7 4 11 0 CO N7 8 7 0 F8 8 7 0 CON8 2 12 1 F9 10 5 0 CON9 4 11 0 F10 8 7 0 CON10 13 2 0 TOTAL 38 83 26 TOTAL 70 76 4 Session 11 Subject Avoids Escapes Failures Subject Avoids Escapes Failures SB1 0 8 7 FSB1 3 4 8 SB2 7 8 0 FSB2 6 7 2 SB3 4 11 0 FSB3 5 8 2 SB4 8 6 1 FSB4 4 10 1 SB5 2 13 0 FSB5 5 10 0 SB6 10 5 0 FSB6 3 11 1 SB7 0 15 0 FSB7 12 3 0 SB8 4 4 7 FSB8 0 4 11 SB9 8 6 1 FSB9 8 7 0

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70 SB10 8 7 0 FSB10 4 11 0 TOTAL 51 83 16 TOTAL 50 75 25 Subject Avoids Escapes Failures Subject Avoid s Escapes Failures F1 1 14 0 CON1 7 8 0 F2 2 9 4 CON2 9 6 0 F3 6 9 0 CON3 0 15 0 F4 0 13 2 CON4 12 3 0 F5 0 3 12 CON5 7 8 0 F6 0 4 11 CON6 6 9 0 F7 6 9 0 CON7 8 7 0 F8 7 8 0 CON8 5 10 0 F9 8 7 0 CON9 5 10 0 F10 10 5 0 CON10 8 7 0 TOTAL 40 81 29 TOTAL 67 83 0 Session 12 Subject Avoids Escapes Failures Subject Avoids Escapes Failures SB1 0 7 8 FSB1 0 3 12 SB2 11 4 0 FSB2 5 8 2 SB3 5 10 0 FSB3 5 8 2 SB4 9 6 0 FSB4 2 13 0 SB5 3 12 0 FSB5 9 6 0 SB6 8 7 0 FSB6 3 12 0 SB7 2 13 0 FSB7 9 6 0 SB8 3 6 6 FSB8 1 7 7 SB9 11 4 0 FSB9 12 3 0 SB10 7 8 0 FSB10 3 12 0 TOTAL 59 77 14 TOTAL 49 78 23 Subject Avoids Escapes Failures Subject Avoids Escapes Failures F1 0 15 0 CON1 9 6 0 F2 1 8 6 CON2 5 10 0 F3 6 9 0 CON3 0 15 0 F4 0 7 8 CON4 12 3 0 F5 0 5 10 CON5 9 6 0

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71 F6 0 6 9 CON6 9 6 0 F7 9 6 0 CON7 9 6 0 F8 6 9 0 CON8 3 12 0 F9 14 1 0 CON9 8 7 0 F10 12 3 0 CON10 11 4 0 TOTAL 48 69 33 TOTAL 75 75 0 Session 13 Subject Avoids Escapes Failures Subject Avoids E scapes Failures SB1 0 8 7 FSB1 1 5 9 SB2 9 6 0 FSB2 6 9 0 SB3 6 9 0 FSB3 5 9 1 SB4 11 4 0 FSB4 0 15 0 SB5 3 10 2 FSB5 5 9 1 SB6 9 6 0 FSB6 1 14 0 SB7 2 11 2 FSB7 8 7 0 SB8 2 11 2 FSB8 0 6 9 SB9 7 8 0 FSB9 12 3 0 SB10 6 7 2 FSB10 6 9 0 TOTAL 55 80 15 TOTAL 44 86 20 Subject Avoids Escapes Failures Subject Avoids Escapes Failures F1 1 13 1 CON1 10 5 0 F2 2 12 1 CON2 9 6 0 F3 6 9 0 CON3 0 14 1 F4 0 13 2 CON4 13 2 0 F5 0 2 13 CON5 12 3 0 F6 4 5 6 CON6 6 9 0 F7 0 13 2 CON7 5 9 0 F8 4 11 0 CON8 2 13 0 F9 10 5 0 CON9 10 5 0 F10 12 3 0 CON10 12 3 0 TOTAL 39 86 25 TOTAL 79 69 0 Session 14

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72 Subject Avoids Escapes Failures Subject Avoids Escapes Failures SB1 0 7 8 FSB1 3 5 7 SB2 9 6 0 FSB2 8 7 0 SB3 7 8 0 FSB3 5 9 1 SB4 8 7 0 FSB4 5 10 0 SB5 4 11 0 FSB5 8 7 0 SB6 6 7 2 FSB6 2 12 1 SB7 2 12 1 FSB7 4 11 0 SB8 4 8 3 FSB8 1 6 8 SB9 11 4 0 FSB9 11 4 0 SB10 6 7 2 FSB10 4 11 0 TOTAL 57 77 16 TOTAL 51 82 17 Subject Avoids Escapes Failures Subject Avoids Es capes Failures F1 0 12 3 CON1 11 4 0 F2 6 6 3 CON2 7 8 0 F3 8 7 0 CON3 1 14 0 F4 0 14 1 CON4 12 3 0 F5 0 4 11 CON5 7 8 0 F6 3 6 6 CON6 7 8 0 F7 4 8 3 CON7 12 3 0 F8 4 11 0 CON8 1 11 3 F9 8 7 0 CON9 11 4 0 F10 8 7 0 CON10 10 5 0 TOTAL 4 1 82 27 TOTAL 79 68 3 Session 15 Subject Avoids Escapes Failures Subject Avoids Escapes Failures SB1 1 3 11 FSB1 3 6 6 SB2 9 6 0 FSB2 8 6 1 SB3 7 8 0 FSB3 5 9 1 SB4 7 8 0 FSB4 2 13 0 SB5 4 11 0 FSB5 5 9 1 SB6 6 7 2 FSB6 2 13 0 SB7 1 14 0 FSB7 5 10 0 SB8 4 6 5 FSB8 0 13 2 SB9 8 7 0 FSB9 11 4 0

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73 SB10 6 7 2 FSB10 2 13 0 TOTAL 53 77 20 TOTAL 43 96 11 Subject Avoids Escapes Failures Subject Avoids Escapes Failures F1 0 13 2 CON1 12 3 0 F2 1 8 6 CON2 11 4 0 F3 4 11 0 CON3 0 14 1 F4 2 12 1 CON4 9 6 0 F5 0 3 12 CON5 11 4 0 F6 1 8 6 CON6 13 2 0 F7 4 8 3 CON7 7 8 0 F8 6 9 0 CON8 5 10 0 F9 11 4 0 CON9 13 2 0 F10 12 3 0 CON10 12 3 0 TOTAL 41 79 30 TOTAL 93 56 1 Session 16 Subject Avoids Escapes Failures Subject Avoids Escapes Failures SB1 0 6 9 FSB1 3 8 4 SB2 12 3 0 FSB2 11 4 0 SB3 5 10 0 FSB3 5 9 1 SB4 4 11 0 FSB4 3 11 1 SB5 9 6 0 FSB5 5 9 1 SB6 6 7 2 FSB6 0 15 0 SB7 7 8 0 FSB7 4 10 1 SB8 2 9 4 FSB8 3 5 7 SB9 8 7 0 FSB9 7 8 0 SB10 6 7 2 FSB10 6 9 0 TOTAL 59 74 17 TOTAL 47 88 15 Subject Avoids Escapes Failures Subject Avoids Escapes Failures F1 1 10 4 CON1 9 6 0 F2 3 11 1 CON2 7 8 0 F3 7 8 0 CON3 2 13 0 F4 0 12 3 CON4 11 4 0 F5 0 3 12 CON5 11 4 0

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74 F6 1 9 5 CON6 3 12 0 F7 4 8 3 CON7 6 9 0 F8 9 6 0 CON8 1 14 0 F9 11 4 0 CON9 13 2 0 F10 3 12 0 CON10 11 4 0 TOTAL 39 83 28 TOTAL 74 76 0 Avoidance and Escape Latency The following lists all raw latency data for all subjects in order of second of stimulus. Session 5 latency is list ed first with SB and FSB groups followed by F and CON groups. Session 16 latency is listed second with the same group order Data marked with an asterisk indicates dummy data, used to fill gaps with missing data due to subject death, equipment malfunction etc. Session 5 Second Subject Avoid Escape Second Subject Avoid Escape 1 SB1 0 8 1 FSB1 0 1 1 SB2 0 3 1 FSB2 0 4 1 SB3 0 5 1 FSB3 0 1 1 SB4 0 7 1 FSB4 0 6 1 SB5 0 4 1 FSB5 0 3 1 SB6 0 5 1 FSB6 0 8 1 SB7 0 8 1 FSB7 0 6 1 SB8 0 7 1 FSB8 0 2 1 SB9 0 2 1 FSB9 0 3 1 SB10 0 6 1 FSB10 0 9 2 SB1 0 2 2 FSB1 0 3 2 SB2 2 0 2 FSB2 0 6 2 SB3 0 4 2 FSB3 0 3 2 SB4 0 1 2 FSB4 0 5 2 SB5 0 2 2 FSB5 0 5 2 SB6 0 2 2 FSB6 0 2 2 SB7 0 2 2 FSB7 0 3 2 SB8 0 5 2 FSB8 0 1

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75 2 SB9 1 2 2 FSB9 0 1 2 SB10 0 4 2 FSB10 0 2 3 SB1 0 4 3 FSB1 0 2 3 SB2 2 2 3 FSB2 0 2 3 SB3 1 2 3 FSB3 1 0 3 SB4 0 0 3 FSB4 0 1 3 SB5 0 3 3 FSB5 0 1 3 SB6 0 1 3 FSB6 0 3 3 SB7 0 1 3 FSB7 0 1 3 SB8 0 0 3 FSB8 0 3 3 SB9 1 2 3 FSB9 1 0 3 SB10 0 2 3 FSB10 1 2 4 SB1 0 0 4 FSB1 0 2 4 SB2 1 0 4 FSB2 0 2 4 SB3 0 0 4 FSB3 3 2 4 SB4 1 0 4 FSB4 0 0 4 SB5 0 0 4 FSB5 1 0 4 SB6 0 0 4 FSB6 0 1 4 SB7 0 1 4 FSB7 1 2 4 SB8 0 0 4 FSB8 0 0 4 SB9 1 0 4 FSB9 1 1 4 SB10 0 0 4 FSB10 0 1 5 SB1 0 0 5 FSB1 0 0 5 SB2 1 0 5 FSB2 0 0 5 SB3 0 0 5 FSB3 0 1 5 SB4 1 0 5 FSB4 0 0 5 SB5 0 2 5 FSB5 2 0 5 SB6 1 0 5 FSB6 0 1 5 SB7 0 1 5 FSB7 0 1 5 SB8 1 0 5 FSB8 0 0 5 SB9 3 0 5 FSB9 2 0 5 SB10 0 1 5 FSB10 0 0 6 SB1 1 6 FSB1 0 6 SB2 1 6 FSB2 0 6 S B3 1 6 FSB3 1 6 SB4 2 6 FSB4 0 6 SB5 0 6 FSB5 0 6 SB6 1 6 FSB6 0 6 SB7 0 6 FSB7 1 6 SB8 0 6 FSB8 0 6 SB9 0 6 FSB9 2 6 SB10 0 6 FSB10 0

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76 7 SB1 0 7 FSB1 0 7 SB2 1 7 FSB2 0 7 SB3 0 7 FSB3 0 7 SB4 1 7 FSB4 0 7 SB5 1 7 FSB5 1 7 SB6 2 7 FSB6 0 7 SB7 0 7 FSB7 0 7 SB8 0 7 FSB8 0 7 SB9 2 7 FSB9 2 7 SB10 1 7 FSB10 0 8 SB1 0 8 FSB1 1 8 SB2 2 8 FSB2 0 8 SB3 1 8 FSB3 0 8 SB4 1 8 FSB4 1 8 SB5 1 8 FSB5 1 8 SB6 2 8 FSB6 0 8 SB7 0 8 FSB7 0 8 SB8 0 8 FSB8 0 8 SB9 1 8 FSB9 0 8 SB10 0 8 FSB10 0 9 SB1 0 9 FSB1 0 9 SB2 0 9 FSB2 0 9 SB3 0 9 FSB3 2 9 SB4 0 9 FSB4 0 9 SB5 0 9 FSB5 1 9 SB6 0 9 FSB6 0 9 SB7 0 9 FSB7 0 9 SB8 1 9 FSB8 0 9 SB9 0 9 FSB9 1 9 SB10 1 9 FSB10 0 10 SB1 0 10 FSB1 0 10 SB2 0 10 FSB2 0 10 SB3 1 10 FSB3 0 10 SB4 1 10 FSB4 1 10 SB5 0 10 FSB5 0 10 SB6 1 10 FSB6 0 10 SB7 0 10 FSB7 0 10 SB8 1 10 FSB8 0 10 SB9 0 10 FSB9 0 10 SB10 0 10 FSB10 0

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77 1 F1 0 10 1 CON1 0 2 1 F2 0 2 1 CON2 0 5 1 F3 0 10 1 CON3 0 2 1 F4 0 3 1 CON4 0 7 1 F5 0 7 1 CON5 0 8 1 F6 0 2 1 CON6 0 3 1 F7 0 4 1 CON7 0 6 1 F8 0 8 1 CON8 0 10 1 F9 0 4 1 CON9 0 9 1 F10 0 6 1 CON10 0 5 2 F1 0 3 2 CON1 0 2 2 F2 0 3 2 CON2 0 0 2 F3 1 1 2 CON3 0 4 2 F4 0 3 2 CON4 0 2 2 F5 0 3 2 CON5 1 3 2 F6 0 1 2 CON6 1 2 2 F7 0 3 2 CON7 0 1 2 F8 0 5 2 CON8 0 3 2 F9 0 0 2 CON9 0 2 2 F10 0 7 2 CON10 0 2 3 F1 0 2 3 CON1 5 1 3 F2 0 3 3 CON2 2 0 3 F3 0 1 3 CON3 1 4 3 F4 0 1 3 CON4 0 1 3 F5 0 1 3 CON5 1 0 3 F6 0 0 3 CON6 1 1 3 F7 0 1 3 CON7 5 1 3 F8 0 1 3 CON8 0 0 3 F9 2 1 3 CON9 0 1 3 F10 0 0 3 CON10 0 0 4 F1 0 0 4 CON1 0 0 4 F2 0 1 4 CON2 2 0 4 F3 1 0 4 CON3 0 2 4 F4 0 0 4 CON4 2 0 4 F5 0 0 4 CON5 0 0 4 F6 0 0 4 CON6 2 0 4 F7 2 1 4 CON7 0 0 4 F8 0 0 4 CON8 0 0 4 F9 1 0 4 CON9 0 2 4 F10 0 1 4 CON10 0 0 5 F1 0 0 5 CON1 1 0 5 F2 0 1 5 CON2 1 0

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78 5 F3 1 0 5 CON3 0 0 5 F4 1 0 5 CON4 0 0 5 F5 0 2 5 CON5 0 0 5 F6 0 0 5 CON6 1 0 5 F7 0 0 5 CON7 1 1 5 F8 0 1 5 CON8 0 1 5 F9 2 0 5 CON9 0 0 5 F10 0 0 5 CON10 0 1 6 F1 0 6 CON1 0 6 F2 0 6 CON2 2 6 F3 0 6 CON3 0 6 F4 1 6 CON4 1 6 F5 0 6 CON5 0 6 F6 0 6 CON6 1 6 F7 0 6 CON7 0 6 F8 0 6 CON8 0 6 F9 0 6 CON9 0 6 F10 0 6 CON10 0 7 F1 0 7 CON1 3 7 F2 0 7 CON2 0 7 F3 0 7 CON3 0 7 F4 3 7 CON4 1 7 F5 0 7 CON5 1 7 F6 0 7 CON6 0 7 F7 1 7 CON7 0 7 F8 0 7 CON8 0 7 F9 2 7 CON9 0 7 F10 0 7 CON10 2 8 F1 0 8 CON1 0 8 F2 0 8 CON2 0 8 F3 0 8 CON3 0 8 F4 0 8 CON4 0 8 F5 0 8 CON5 1 8 F6 0 8 CON6 1 8 F7 2 8 CON7 0 8 F8 0 8 CON8 0 8 F9 2 8 CON9 0 8 F10 1 8 CON10 2 9 F1 0 9 CON1 1 9 F2 0 9 CON2 1 9 F3 0 9 CON3 0 9 F4 2 9 CON4 0

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79 9 F5 0 9 CON5 0 9 F6 0 9 CON6 1 9 F7 1 9 CON7 0 9 F8 0 9 CON8 0 9 F9 1 9 CON9 0 9 F10 0 9 CON10 1 10 F1 0 10 CON1 0 10 F2 0 10 CON2 2 10 F3 0 10 CON3 0 10 F4 0 10 CON4 1 10 F5 0 10 CON5 0 10 F6 0 10 CON6 1 10 F7 0 10 CON7 0 10 F8 0 10 CON8 0 10 F9 0 10 CON9 1 10 F10 0 10 CON10 0 Session 16 Second Subject Avoid Escape Second Subject Avoid Escape 1 SB1 0 2 1 FSB1 0 1 1 SB2 0 1 1 FSB2 0 3 1 SB3 0 6 1 FSB3 0 5 1 SB4 0 11 1 FSB4 0 6 1 SB5 0 2 1 FSB5 0 6 1 SB6 0 3 1 FSB6 0 10 1 SB7 0 1 1 FSB7 0 5 1 SB8 0 4 1 FSB8 0 0 1 SB9 0 2 1 FSB9 0 5 1 SB10 0 3 1 FSB10 0 4 2 SB1 0 0 2 FSB1 0 0 2 SB2 1 2 2 FSB2 0 0 2 SB3 0 3 2 FSB3 0 2 2 SB4 0 0 2 FSB4 0 4 2 SB5 0 4 2 FSB5 0 1 2 SB6 0 3 2 FSB6 0 3 2 SB7 0 5 2 FSB7 0 3 2 SB8 0 4 2 FSB8 0 0 2 SB9 1 3 2 FSB9 0 2 2 SB10 0 3 2 FSB10 0 2

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80 3 SB1 0 1 3 FSB1 0 3 3 SB2 2 0 3 FSB2 0 1 3 SB3 1 1 3 FSB3 0 2 3 SB4 0 0 3 FSB4 0 0 3 SB5 1 0 3 FSB5 2 2 3 SB6 1 1 3 FSB6 0 1 3 SB7 1 2 3 FSB7 0 0 3 SB8 0 0 3 FSB8 0 4 3 SB9 0 1 3 FSB9 0 1 3 SB10 1 1 3 FSB10 0 2 4 SB1 0 2 4 FSB1 0 3 4 SB2 3 0 4 FSB2 1 0 4 SB3 0 0 4 FSB3 0 1 4 SB4 0 0 4 FSB4 0 1 4 SB5 1 0 4 FSB5 1 0 4 SB6 1 0 4 FSB6 0 1 4 SB7 1 0 4 FSB7 0 1 4 SB8 0 1 4 FSB8 0 0 4 SB9 0 0 4 FSB9 1 0 4 SB10 1 0 4 FSB10 0 0 5 SB1 0 1 5 FSB1 1 1 5 SB2 1 0 5 FSB2 2 0 5 SB3 0 0 5 FSB3 1 0 5 SB4 0 0 5 FSB4 0 0 5 SB5 0 0 5 FSB5 0 0 5 SB6 0 0 5 FSB6 0 0 5 SB7 0 0 5 FSB7 0 1 5 SB8 1 0 5 FSB8 1 1 5 SB9 0 1 5 FSB9 1 0 5 SB10 0 0 5 FSB10 0 1 6 SB1 0 6 FSB1 0 6 SB2 4 6 FSB2 1 6 SB3 0 6 FSB3 0 6 SB4 1 6 FSB4 0 6 SB5 3 6 FSB5 0 6 SB6 1 6 FSB6 0 6 SB7 0 6 FSB7 0 6 SB8 0 6 FSB8 0 6 SB9 0 6 FSB9 1 6 SB10 1 6 FSB10 1 7 SB1 0 7 FSB1 0 7 SB2 1 7 FSB2 0

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81 7 SB3 2 7 FSB3 1 7 SB4 0 7 FSB4 0 7 SB5 2 7 FSB5 0 7 SB6 1 7 FSB6 0 7 SB7 1 7 FSB7 1 7 SB8 0 7 FSB8 1 7 SB9 2 7 FSB9 0 7 SB10 1 7 FSB10 2 8 SB1 0 8 FSB1 1 8 SB2 0 8 FSB2 2 8 SB3 0 8 FSB3 1 8 SB4 1 8 FSB4 0 8 SB5 0 8 FSB5 0 8 SB6 0 8 FSB6 0 8 SB7 0 8 FSB7 0 8 SB8 0 8 FSB8 0 8 SB9 0 8 FSB9 2 8 SB10 0 8 FSB10 0 9 SB1 0 9 FSB1 1 9 SB2 0 9 FSB2 3 9 SB3 1 9 FSB3 1 9 SB4 0 9 FSB4 2 9 SB5 1 9 FSB5 1 9 SB6 1 9 FSB6 0 9 SB7 2 9 FSB7 0 9 SB8 0 9 FSB8 0 9 SB9 4 9 FSB9 2 9 SB10 1 9 FSB10 2 10 SB1 0 10 FSB1 0 10 SB2 0 10 FSB2 2 10 SB3 1 10 FSB3 1 10 SB4 2 10 FSB4 1 10 SB5 1 10 FSB5 1 10 SB6 1 10 FSB6 0 10 SB7 2 10 FSB7 3 10 SB8 1 10 FSB8 1 10 SB9 1 10 FSB9 0 10 SB10 1 10 FSB10 1 1 F1 0 7 1 CON1 0 5

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82 1 F2 0 1 1 CON2 0 5 1 F3 0 5 1 CON3 0 8 1 F4 0 6 1 CON4 0 4 1 F5 0 0 1 CON5 0 4 1 F6 0 3 1 CON6 0 9 1 F7 0 3 1 CON7 0 3 1 F8 0 3 1 CON8 0 8 1 F9 0 0 1 CON9 0 2 1 F10 0 9 1 CON10 0 3 2 F1 0 1 2 CON1 0 1 2 F2 0 3 2 CON2 0 1 2 F3 1 2 2 CON3 0 2 2 F4 0 3 2 CON4 0 0 2 F5 0 1 2 CON5 0 0 2 F6 0 1 2 CON6 0 2 2 F7 0 2 2 CON7 0 5 2 F8 0 2 2 CON8 0 4 2 F9 0 4 2 CON9 0 0 2 F10 0 3 2 CON10 1 1 3 F1 0 2 3 CON1 1 0 3 F2 0 3 3 CON2 0 2 3 F3 2 1 3 CON3 0 1 3 F4 0 2 3 CON4 1 0 3 F5 0 1 3 CON5 0 0 3 F6 0 2 3 CON6 0 1 3 F7 1 1 3 CON7 0 1 3 F8 0 0 3 CON8 0 1 3 F9 4 0 3 CON9 0 0 3 F10 0 0 3 CON10 2 0 4 F1 0 0 4 CON1 1 0 4 F2 0 2 4 CON2 0 0 4 F3 0 0 4 CON3 0 2 4 F4 0 0 4 CON4 1 0 4 F5 0 1 4 CON5 0 0 4 F6 0 1 4 CON6 0 0 4 F7 0 1 4 CON7 1 0 4 F8 1 1 4 CON8 0 1 4 F9 0 0 4 CON9 1 0 4 F10 3 0 4 CON10 0 0 5 F1 0 0 5 CON1 0 0 5 F2 0 2 5 CON2 0 0 5 F3 2 0 5 CON3 0 0

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83 5 F4 0 1 5 CON4 1 0 5 F5 0 0 5 CON5 1 0 5 F6 0 2 5 CON6 0 0 5 F7 0 1 5 CON7 0 0 5 F8 0 0 5 CON8 0 0 5 F9 1 0 5 CON9 5 0 5 F10 0 0 5 CON10 1 0 6 F1 0 6 CON1 1 6 F2 0 6 CON2 0 6 F3 0 6 CON 3 0 6 F4 0 6 CON4 3 6 F5 0 6 CON5 2 6 F6 0 6 CON6 0 6 F7 0 6 CON7 3 6 F8 2 6 CON8 0 6 F9 0 6 CON9 2 6 F10 0 6 CON10 1 7 F1 0 7 CON1 2 7 F2 0 7 CON2 0 7 F3 1 7 CON3 0 7 F4 0 7 CON4 2 7 F5 0 7 CON5 4 7 F6 0 7 CON6 1 7 F7 1 7 CON7 0 7 F8 2 7 CON8 0 7 F9 2 7 CON9 3 7 F10 0 7 CON10 4 8 F1 0 8 CON1 1 8 F2 1 8 CON2 0 8 F3 0 8 CON3 0 8 F4 0 8 CON4 1 8 F5 0 8 CON5 1 8 F6 0 8 CON6 0 8 F7 1 8 CON7 0 8 F8 1 8 CON8 0 8 F9 3 8 CON9 1 8 F10 0 8 CON10 1 9 F1 0 9 CON1 2 9 F2 1 9 CON2 2 9 F3 1 9 CON3 0 9 F4 0 9 CON4 1 9 F5 0 9 CON5 2

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84 9 F6 0 9 CON6 1 9 F7 1 9 CON7 2 9 F8 3 9 CON8 1 9 F9 0 9 CON9 1 9 F10 0 9 CON10 0 10 F1 1 10 C ON1 1 10 F2 1 10 CON2 5 10 F3 0 10 CON3 2 10 F4 0 10 CON4 1 10 F5 0 10 CON5 1 10 F6 1 10 CON6 1 10 F7 0 10 CON7 0 10 F8 0 10 CON8 0 10 F9 1 10 CON9 0 10 F10 0 10 CON10 1

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85 Appendix B: Subject Deaths and Anomalies Subj ect Deaths The following is a complete list of all subjects which died during the experiment, which Session Day they died, and which Session Day their replacement was introduced (if applicable). Days listed refer to death or replacement prior to running of sessions that day. Subject Deaths Subject Death Day Replacement Day SB1 7 9 SB5 5 6 SB6 15 Not Replaced SB7 6 9 SB8 8 9 SB10 13 Not Replaced FSB1 4 6 FSB3 11 Not Replaced FSB7 6 9 FSB8 3 6 F2 6 9 F 5 8 9 F6 5 6 F7 14 Not Replaced CON3 1 6 CON6 3 6 CON8 Water Change 9 CON10 6 9

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86 Other Anomalies The following is a complete list of occurrences which prevented data from being collected the subjects which were affected, a nd the number of times in which they occurred. Subject # of Occurrences Description of Anomaly SB6 1 Shock delivery disconnected SB8 1 Shock delivery disconnected FSB5 5 Jumped out of shuttle box mid session FSB9 1 Shuttle box not r ecording crossovers F4 2 Shuttle box not recording crossovers F6 1 Shuttle box not recording crossovers F8 1 Shuttle box not recording crossovers F9 1 Shuttle box not recording crossovers F10 1 Shuttle box not recording crossovers

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87 A ppendix C: Programming Methodology The following details the complete programming of Graphic State Notation TM and the step by step protocol of the shuttle box avoidance paradigm. An experimental session has 4 programmed states: State 1: Lights off, Shock O ff. This is the starting condition for each trial. Both stimuli (the lights and the shock) are turned off. State 1 is set to run for 30 seconds, at which point a randomization check is performed. There is then a 52% chance of entering State 2. If State 2 i s not entered, the 30 second clock is reset and the process repeats until State 2 is entered. This randomization of the duration of State 1 removes the element of time as a variable, ensuring that the subjects behaviors are based the stimuli and not the pa ssage of time. Tripping of the photo sensor is not recorded during this state. After entering this state 16 times (after 15 trials) the program will transition to Fin, ending the session. State 2: Lights on, Shock off. At the beginning of this state, the c onditioned stimulus (the light) is turned on and will remain on for the duration of this state. Any tripping of the photo sensor during this state will be recorded as an "avoid" (the subject responded to the conditional stimulus and successfully avoided th e unconditional stimulus) and the program will immediately revert to State 1. This state is set to last for 10 seconds unless the photo sensor is tripped. If the photo sensor is not tripped during this state, the program will transition to State 3.

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88 State 3 : Lights on, Shock on. As soon as this state is entered, the unconditional stimulus is activated. A 3.8 V pulsed shock is delivered at a rate of 1 Hz with a pulse width of 500 ms. Any tripping of the photo sensor during this state will be recorded as an "e scape" (the subject escaped from the unconditional stimulus) and the program will immediately revert to State 1.This state is set to last for 5 seconds and if the photo sensor is not tripped during this interval, the program will enter State 4. State 4: Li ghts off, Shock off. Entering of this state is recorded as a "failure" (the subject failed to respond to either stimulus). This state is set to last for 3 seconds before returning to State 1. Tripping of the photo sensor is not recorded during this state. Fin: This state is entered after 15 trials and ends the session.

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