This item is only available as the following downloads:
i THE RETURN OF FISH ON PROZAC : FURTHER STUDIES INTO SEROTONERGIC MANIPULATION AND AVOIDANCE LEARNING IN GOLDFISH ( CARASSIUS AURATUS ). Title BY ALEXANDER THOMAS HIBBERD A Thesis Submitted to the Division of Natural Science New College of Florida in partial fulfillment of the requirements for the degree Bachelor of Arts Under the sponsorship of Dr. Alfred Beulig Sarasota, Florida March, 2010
i i Acknowledgements I would like to thank my s ponsor, Dr. Alfred Beulig, for his enthusiasm, support, and everlasting patience, as well as my committee members, Dr. Leo Demski and Dr. Gordon Bauer. Additional thanks go out to Joel Beaver, who helped me acquire my subjects and keep them alive, and to my friends and family members, for their emotion al and intellectual support and interest. Finally, I would like to give a special thanks to all sixty five fish who participated in t he study without whom this experiment would not have been possible
iii Table of Contents TITLE ................................ ................................ ................................ ................................ ............................. I ACKNOWLEDGEMENTS ................................ ................................ ................................ ......................... II TABLE OF CONTENTS ................................ ................................ ................................ ........................... III ABSTRACT ................................ ................................ ................................ ................................ ................ IV HISTORICAL OVERVIEW OF THE DISCOVERY OF SEROTONIN AND ANTI DEPRESSANT MEDICATIONS. ................................ ................................ ................................ ................................ ........... 1 NEUROANATOMICAL OVER VIEW OF THE FUNCTION AND LOCATIONS OF SER OTONIN IN THE HUMAN BRAIN. ................................ ................................ ................................ ............................ 4 PHYLOGENY OF SEROTON IN AS A NEUROTRANSMI TTER. ................................ ........................ 6 THE EVOLUTIONARY HIS TORY OF TELEOSTEI. ................................ ................................ ............ 8 NEUROPHYSIOLOGICAL M ECHANISMS OF SEROTON IN. ................................ ......................... 10 C HEMICAL MECHANISMS O F SEROTONIN ................................ ................................ ................................ ..... 10 N EUROLOGICAL MECHANIS MS OF SEROTONIN RECE PTORS ................................ ................................ ........... 12 F UNCTIONS OF 5 HT1 A AND 5 HT1 B. ................................ ................................ ................................ .......... 14 N EUROLOGICAL MECHANIS M S ................................ ................................ ................................ .......... 16 SEROTONIN, BEHAVIOR, AND TELEOSTI. ................................ ................................ ....................... 16 RATIONALE FOR CURREN T EXPERIMENT. ................................ ................................ .................... 17 WAY 100,635 ................................ ................................ ................................ ................................ ............ 18 F LUOXETINE ................................ ................................ ................................ ................................ .............. 20 C O A DMINISTRATION OF WAY 100,635 AND F LUOXETINE ................................ ................................ .......... 22 M ATERIALS AND M ETHODS ................................ ................................ ................................ ................... 22 S UBJECTS ................................ ................................ ................................ ................................ .................. 23 H OME A QUARIA ................................ ................................ ................................ ................................ ........ 24 M AIN TENANCE AND F EEDING ................................ ................................ ................................ ..................... 25 S HUTTLE B OX A QUARIA ................................ ................................ ................................ ............................. 26 C OLBOURN I NSTRUMENTS G RAPHIC S TATE N OTATION ................................ ................................ ............ 28 D RUG A DMINISTRATION ................................ ................................ ................................ ............................. 29 S UBJECT A SSIGNMENT DEATHS AND ANOMALIES ................................ ................................ ....................... 33 E XPERIMENTAL P ROTOCOL ................................ ................................ ................................ ......................... 34 RESULTS. ................................ ................................ ................................ ................................ .................... 35 A VOIDANCE R ESPONSES ................................ ................................ ................................ ............................. 35 F AILURES ................................ ................................ ................................ ................................ .................. 39 E SCAPES ................................ ................................ ................................ ................................ ................... 43 P ERFORMANCE V ARIABILITY ................................ ................................ ................................ ...................... 44 C OMPARISONS BETWEEN G ROUPS ................................ ................................ ................................ ............... 46 S UPPORT OF H YPOTHESES AND C ONCLUSIONS ................................ ................................ ........................... 48 REFERENCES ................................ ................................ ................................ ................................ ........... 63
iv THE RETURN OF FISH ON PROZAC: FURTHER STUDIES INTO SEROTONERGIC MANIPULATION AND AV OIDANCE LEARNING IN GOLDFISH ( CARASSIUS AURATUS ). Alex Hibberd New College of Florida, 2010 A BSTRACT In a continuation of previous work to confirm the possible evolutionary conservation of serotonergic mechanisms in teleosts as compared to other chordate groups, behavioral responses to serotonin manipulation were examined in the common goldfish, Carassius auratus when subjected to a two way active avoidance learning (shuttle box) model. A previous study of a similar nature failed to find expecte d significant differences between fish given fluoxetine (Prozac), an SSRI intended to potentiate serotonergic transmission, and an untreated control group. To determine if this result was due to a dose dependent mechanism, the experiment was repeated with double dosages administered. Additionally, WAY 100,635, a selective serotonin receptor antagonist was administered to another group to confirm that inhibition of serotonergic transmission results in increased avoidance performance in goldfish. Finally, a c ombination group of both drugs was tested to determine what, if any, possible interactions between treatments occur when co administered.
v As expected, the WAY group performed better than other groups in the avoidance task, while the fluoxetine group unde r performed in comparison to controls. Neither group showed statistically significant difference from control group due to unexpectedly high subject response variability. The co administration group receiving both WAY and fluoxetine performed very poorly i n comparison to all other groups, with a statistically significant difference between this group and WAY group (P < .05). These results support the conclusions of the previous experiment, as well as shed light on an interesting and potentially novel intera ction effect between fluoxetine and WAY in teleosts. Dr. Alfred Beulig Division of Natural Sciences
1 Historical overview of the discovery of serotonin and anti depressant medications. The first research that led to the eventual d iscovery and isolation of serotonin began not from neuropsychological or neurochemical research, but rather, work conducted with the goal of discovering causes and treatment for chronic hypertension, and was discovered independently by two groups. In the r esearch into the properties of amines found to cause muscle constriction, derived from the viscera of several species. One of the substances discovered was found in gut enterochroma ffin cells. Erspamer noticed that a concentration of this substance caused smooth muscle contraction in the uterus of rats, and he named it enteramine (Whitaker Azmitia, 1999). sim ilar muscle contraction properties on the small intestine of rabbits, dog bladders, and the hearts of several mollusks. This work came to a head in 1952 after enteramine was isolated by processing more than thirty kilograms of salivary tissue taken from th irty thousand specimens of Octopus Vulgaris By immersing the salivary glands in acetone and draining the supernatant, and then washing and recrystalizing with various solvents to remove impurities, enough enteramine was obtained to identify the chemical s tructure: 5 hydroxytryptamine (5 HT). It was at this time that Erspamer realized that enteramine was identical in chemical composition and pharmacological effect to serotonin, a chemical that had recently been isolated in 1949 by American scientists (Erspa mer, Asero, 1953).
2 Meanwhile, the aforementioned group of American scientists, Drs. Page, Green, and Rapport, were doing their own investigations into possible chemical causes of hypertension. Page believed that hypertension was the result of renin. Howeve r, in attempting to isolate renin from the blood, he repeatedly found his efforts blocked by an unknown substance he called a serum vasoconstrictor (Page, 1968). Using serum prepared from beef blood, these scientists were able to prepare assays though whic h they also isolated 5 HT, which they named serotonin, due to isolating it from serum (Rapport, et al. 1948a, Rapport, et al. 1948b, Rapport, et al. 1948c). Finally, in 1953, papers about serotonin and enteramine came to the attention of a scientist named Dr. Betty Twarog, who noticed that the chemical had most of the same structural requirements as a neurotransmitter she postulated to exist in conjunction with acetylcholine in mussels (Whitaker Azmitia, 1999). Working with Dr. Page, she tested and measure d serotonin activity in various tissues, blood, and urine in dogs, rabbits, and rats, and discovered that the chemical was always present in several areas of the brains of rather than a vasoconstrictor (Twarog, Page, 1953). From this point, serotonin quickly became a major topic of research in relation to the chemistry of the brain, mental illness, and behavior. Early work using lysergic acid diethylamide (LSD) as an antime tabolite of serotonin in muscle tissue suggested that the strong hallucinatory effects and destabilization of mental well being of LSD occurred Shaw, 1954). This hypot hesis was later confirmed when it was found that LSD acts as a
3 5 HT 2a receptor agonist, as well as a partial agonist for several other 5 HT receptors (Nichols, 2004). As the neurochemistry of serotonin and its effects on mood and behavior became better kn own, a tremendous amount of research resulted in developing pharmacological means to modify serotonin levels in the brain to control depression. One of the earliest classes of drug developed to have such an effect is also one of the most powerful: Monoamin e oxidase inhibitors (MAOIs). These drugs work by inhibiting monoamine oxidases, the enzymes that catalyze the oxidation of monoamines, thereby halting or slowing the breakdown of neurotransmitters such as serotonin, epinephrine, dopamine and other neurotr ansmitters. However, the severity of this interaction (which in early MAOIs was often both irreversible and non selective) coupled with the possibility of severe side effects has regulated this class of drug to use ordinarily only in severe cases of atypic al depression (Lenox, Frazer, 2002). serotonin levels had been discovered. These drugs were called selective serotonin reuptake inhibitors (SSRIs) and soon became immensely popular and integrated with modern culture. The basic mechanism of this class of drug is the inhibition of the reuptake of 5 HT by the presynaptic cells, resulting in a larger concentration of 5 HT in the synaptic cleft. The first of these drugs to be patented and made available was called Zimelidine, but has since been widely banned after association with a host of negative side effects, including Guillain Barr syndrome (Carlsson, Wong, 1997). Despite the poor start, the next major drug to be patented, Prozac (Fl uoxetine hydrochloride) soon became commonly used around the world.
4 Drs. Brian Molley and David Rathburn synthesized several derivatives of diphenhydramine, an antihistamine known at the time to have mild anti depressant effects, and discovered nisoxetine a norepinephrine reuptake inhibitor. These results encouraged the testing of similar chemicals for in vitro reuptake of serotonin, the most powerful of which was shown to be fluoxetine (Wong, 1995). Neuroanatomical overview of the function and locations of serotonin in the human brain. The 5 HT system of the human brain is extensive, with most areas of the central nervous system being innervated or affected by serotonergic neurons. Most serotonergic neuron cell bodies are found in the raphe nuclei of the lower brain stem (Page, 1968). These neurons can be divided into caudal and rostral groups (Soares, Gershon, 2000). The rostral subdivision consists primarily of the caudal linear nucleus, the dorsal raphe nucleus (sometimes referred to as the nucleus sup ratrochlearis) and the median raphe nucleus (or superior central raphe nucleus). All of these groups are located within the pons or midbrain (Trk, 2006). The caudal linear nucleus is a small cluster of cell bodies located on the midline of the mesencephal on and contains a mixture of serotonergic and dopaminergic cells. The serotonergic cells of this area are small, round bodied cells, the dendrites of which extend in caudal and rostral directions (Tork, et al, 1984). The dorsal raphe nucleus, located on th prominent component of the rostral serotonergic system. Cell bodies in this area are diverse in size and shape. In the median raphe nucleus, small cells with short dend rites are found on the midline aligned parallel to the mid sagittal plane, while larger cells outside of the midline are not contiguously aligned. (Trk, 2006).
5 The caudal subdivision of the serotonergic system consists of the raphe magnus nucleus, the ra phe pallidus nucleus, and the raphe obscurus nucleus. These areas are found near the midline at the junction of the pons and medulla, between the pyramids of the ventral surface of the medulla, and in a stream extending from the caudal pons to the pyramida l decussation respectively. (Trk, 2006). The projections of both the rostral and caudal subdivisions of brainstem serotonergic cell groups are presented in F ig 1. Fig 1. Schematic of projections of serotonergic cells in the human brain. Obtained from Tork, 2006, unaltered. Originally adapted from Stone, et al 1990.
6 Phylogeny of Serotonin as a Neurotransmitter. As an organic molecule, serotonin is extremely pervasive. Although most frequently thought of as a neurochemical, it serves a plethora of other functions throughout various organic systems (such as working in relation with other chemicals as a vasoconstrictor and vasodilator, as mentioned in previous sections) and is found not only in vertebrates, but invertebrates, plants, and even single celled organisms (Feldman, neurotransmitter, neuromodulator, and neurohormone rather than try to trace its origins beyond the realm of neurochemistry. Serotonin acts as a neuro chemical in species examined from all phyla whose members can be considered to have a nervous system (Weiger, 1997). Ctenophora and Cnidaria, phyla which possess what can arguably be called the simplest nervous systems and which are probably evolutionary o rigins for organized nervous structures, have been shown to use serotonin as a neurochemical in several member species. Immunogold labeling with 5 HT antibodies indicates the presence of serotonin in Aiptasia pallida synaptic vesicles (Westfall et al. 1971 ). Serotonin was detected in interneurons associated with the photoreceptors of cubozoan jellyfish (Martin, 2004). Behavioral experiments have shown that 5 HT has an effect on such things as the amplitude of the rhythmic contractions in sea pansies (Ancti l 1989). Overall, however, investigations into neurotransmission of these phyla are incomplete. (Kass Simon and Paola Pierobon, 2006). In mollusks, the neurological properties of serotonin are better studied. One example is Aplysia californica. In this s pecies, serotonin acts as a neuromodulator to a
7 frequency, and speed are increased. Serotonergic neurons in the cerebral ganglion of this animal facilitate excitatio n of buccal motor neurons (Cheil, et al. 1986). Serotonergic systems have been implicated in other behaviors in several species, including gill and siphon withdrawal reflexes, escape swimming behavior, and sensory response (Weiger, 1997). Leeches have bec ome a model organism for the study of serotonin in annelids. Serotonin containing cells have been shown to be present in all the ventral ganglia of leech species studied. These cells have been shown to employ serotonin as a neurotransmitter, having a stron g impact on feeding behaviors such as swimming, biting, salivation, peristalsis, and mucus secretion. 5 HT has also been shown to have a neurohormonal affect on swimming behavior. (Groome, Clark & Lent, 1993). In arthropods, serotonin has been studied ver y extensively and shown to have complex neurohormonal, neuromodulator, and neurotransmitter roles in species from crustaceans, insects, and arachnids. In this phylum, the level of complexity involved in the serotonergic systems are still less complex than the levels achieved by the vertebrates, and serotonergic cells less frequent, but overall plays a significant role in behavior. (Weiger, 1997). An interesting example of the effect serotonin can have in the behavior of arthropods is an experiment that was done on fruit flies which had their serotonin levels both genetically and pharmacologically reduced: In both, the flies had significant decreases in performance on avoidance tasks involving memory performance (Sitaraman, et al. 2008).
8 The role of seroto nin in the nervous system of chordates is generally more complex than any other phylum. All known vertebrates possess serotonergic neurons in their central nervous system, and the general anatomical location and function of these cells is similar to that a lready discussed in the previous section. It is thus possible to see a general trend in the evolution of serotonergic neurons. In vertebrates, serotonin acts as strongly as a neurotransmitter, and mediates complex behaviors in humans. In the arthropods, serotonin acts as a neurotransmitter, neurohormone, and neruromodulator. Likewise, in leeches, it acts as both a neuromodulator and a neurotransmitter. In gastropods, it acts primarily as a neuromodulator other chemicals are employed as neurotransmitter s ( Weiger, 1997). Similarly, as a survey of body plans and nervous systems decreases in complexity the overall use of serotonin as a neurochemical also becomes less complex, down to the cnidarians and ctenophores, where it promotes the simplest effects, suc h as the modulation of peristaltic contractions (Anctil, 1989). The majority of behavioral and neurological serotonergic research has focused on mammals, many using rats as a model organism. While the rat model provides a valuable and economical means of studying this syste m in mammals, a fish model may prove useful in examining the evolutionary differences between chordates of earlier phylogenetic divergence. The Evolutionary History of Teleostei. The majority of living fish (including goldfish) fall within the teleost infraclass, and more than half of all known vertebrates fall within the category of ray finned fishes (Hurley, 2007).
9 The earliest vertebrate ancestors to these fish are the Agnatha, the jawless fish. The earliest agnathan fossils date back to the lower Ordovician period, between 470 478 million years ago (Erdtmann, et al 2000). As the name implies, these fish are characterized by the lack of bony jaws and teeth. Many of the fossil species found seemed to possess armored bony shields ar ound their heads, unlike modern lampreys and hagfish. One class of these fish, the Osteostraci, is thought to have given rise to the earliest jawed fish before dying out in the late Devonian. They shared several similar characteristics with jawed fish that other classes of Agnatha lack: paired fins, sclerotic bones, and an open endolymphatic duct around the head (Long, 1995). The jawed fish began to rise in the late Ordovician and Silurian periods, and became dominant in the Devonian era (sometimes called acanthodians and placoderms were two of the earliest groups of jawed fish. The placoderms were primarily extant during the Devonian era, and consisted of a large number of species and variation. They are largely considered to be rel ated to the chondrichthyans (which include the Elasmobranchii: s harks, skates, and rays) due to common anatomical specializations such as external reproductive claspers, an eyestalk connecting the eye to the brain case, and similar brain case shape (Long, 1995; Janvier 1997). As far as their evolutionary history is concerned, acanthodians still present some jawed fish that bear a superficial resemblance to sharks in body plan, had an internal skeleton made of cartilage, and died out completely around approximately 250 million
10 years ago. They are considered to be a possible sister group of the osteichthyans., as both groups possess otoliths in the inner ear and similar br ain case shape (Janvier, 1997). Osteichthyans appeared during the late Silurian (410 million years ago) and were well represented in all major groups by the start of the Devonian period. Osteichthyans, the largest and most diverse group of vertebrates, ar e characterized by an ossified internal skeleton marginal teeth on the upper and lower jaw, and pharyngobranchial gill elements on the first two gill arches. They gave rise to the subclass Sarcopterygii (lobed finned fish, which eventually became the first tetrapod land vertebrates) and the subclass Actinopterygii, the ray finned fishes (Long, 1995). The actinopterygians are characterized by the lepidotrichia: fin rays that appear as small bony spines covered by a membrane. This group split into the Chondr ostei and Ceopterygii, which comprise the reedfish, sturgeon, and paddlefish, and the bowfin, and gar, respectively, and the teleosts, which comprise most modern fish species. Teleosts emerged during the Triassic period, and are characterized by a movable maxilla and premaxilla, allowing the jaws to protrude from the mouth, a homocercal caudal fin, and spines which end at the caudal peduncle (Long, 1995; Janvier 1997). Teleosts provide an excellent group of fish from which to choose a model organism, owi ng to the high numbers of modern extant species and the relative ease with which they can be acquired and cared for, in comparison to the lobe finned fish, of which only eight extant species are known, all of which require specialized habitats. Neurophysi ological mechanisms of serotonin. Chemical mechanisms of serotonin.
11 Serotonin is the colloquial term for 5 hydroxytryptamine, with chemical formula C 10 H 12 N 2 O. In most vertebrates, including fish and humans, serotonin is synthesized by brain neurons fr om its chemical precursor, the essential amino acid L tryptophan. Serotonergic neurons utilize two enzymes, tryptophan 5 hydroxylase (TPH) and aromatic L amino acid decarboxylase, the former being unique to serotonergic neurons and the latter found throug hout the brain. TPH catalyzes the rate limiting step of serotonin synthesis, converting L tryptophan to 5 hydroxy L tryptophan, then aromatic L amino acid decarboxylase completes the conversion into serotonin. (Kaufman, 1974). Some serotonin is then conve rted via another pathway to melatonin (N acetyl 5 methoxytryptamine). Dietary intake of tryptophan has been shown to influence serotonin production and levels in the brain (Fernstrom and Wurtman, 1971). For example, rats starved overnight and given a pro tein free diet rich in carbohydrates typically experience an increase in physiological serotonin levels linearly related to the amount of tryptophan consumed. It is thought that the mechanism by which this occurs entails an increase of large neutral amino acids in blood plasma, affecting the rate at which tryptophan is carried across the blood brain barrier by an associated transport mechanism. The corresponding increase in brain tryptophan levels results in an increase in serotonin because TPH is normally unsaturated with substrate, thus, when saturation occurs, both the rate and quantity of serotonin synthesized in the brain increases (Leathwood, 1987). Although acute tryptophan depletion is indicated and used experimentally to reduce serotonin levels, an d has been shown to have subsequent impacts on behaviors such as risk taking (Long, et al. 2009), emotional regulation and memory (Wang, et al.
12 2009, Mendelsohn et al. 2009), and other behaviors thought to be linked to serotonin levels, there is little ind ication that excess dietary tryptophan has a significant impact on normal behavior. In most studies, excess dietary tryptophan only affected behaviors such as appetite and aggression under stress, and only under conditions of high behavioral arousal and su bsequently increased firing of 5 HT neurons (Young, et al. 1988, Stensrud et al.1992, and Chamberlain 1987). In comparison to antidepressant medications, dietary tryptophan supplementation has shown mostly negative or minor results on treating depression o r aggression in psychiatric patients (Young, Leyton, 2002) Neurological mechanisms of serotonin receptors. protein coupled receptors (GPCRs) and ligand gated ion channels that have b een classified as the 5 HT receptors, using a system defined by the International Union of Basic and Clinical Pharmacology. These receptors are further subdivided by family and subtype (see table on following page). A full review of all serotonin receptors is beyond the scope of this document. Instead, attention will be paid mostly to the 5 HT 1A and 5 HT 1B receptors, as these are the most relevant to the proposed study. G protein coupled receptors, including 5 HT 1A and 5 HT 1B are comprised of amino acid sequences unique to each receptor. All, however, contain a sequence of helices that serve as the extracellular binding site. When a ligand binds to the GPCR, it undergoes a conformational change allowing it to activate the associat ed G protein, which may further interact with associated effector enzymes or ion channels. A particular receptor will only activate specific G proteins, which in turn will only activate specific effector systems, resulting in a functional specificity of th e
13 transduction signal (Boess and Martin, 1994). Receptor agonists bind to a receptor, to the receptor and do not affect the receptor itself but instead prevent its use by other ligands. Table 1: Table of 5 HT receptors and function. Generated from information summarized by the IUPHAR database (Hamar, et al. 2009). Receptor Encoding Gene Transducer Implicated Functions or Related Behaviors. 5 HT 1A HTR1A G i /G 0 famil y Addiction. Anxiety. Cardiovascular function. Feeding behavior. Impulsivity. Learning and memory. Respiration. Sexual Behavior. Sleep wakefulness cycles. Vasoconstriction. 5 HT 1B HTR1B G i /G 0 family Addiction. Aggression. Anxiety. Feeding Behavior Learnin g and memory. Sexual Behavior. Vasoconstriction. 5 HT 1D HTR1D G i /G 0 family Anxiety. Locomotion. Vasoconstriction. 5 HT 1E HTR1E G i /G 0 family Unknown. 5 HT 1F HTR1F G i /G 0 family Unknown. 5 HT 2A HTR2A G Q /G 11 family Colonic smooth muscle contraction. Hall ucinogenic mechanisms. Sexual behavior. Sleep wakefulness cycles. Thermoregulation. Vasoconstriction. 5 HT 2B HTR2B G Q /G 11 family Anxiety. Cardiovascular Development. GI contraction. 5 HT 2C HTR2C G Q /G 11 family Anxiety. Feeding behavior. Sleep regulation. Stress response. Obsessive compulsive behavior. 5 HT 3 HTR3A HTR3B Ligand gated ion channel Anxiety. Learning and memory. Locomotion. 5 HT 4 HTR4 G s family Anxiety. Feeding Behavior. Gastric function. Learning and Memory. Respiration. 5 HT 5A HTR5A G i / G 0 family Locomotion. Sleep. 5 HT 6 HTR6 G s family Anxiety. Learning and Memory. 5 HT 7 HTR7 G s family Anxiety. Cardiovascular function. Learning and Memory. Sleep wakefulness cycles. Thermoregulation. Vasoconstriction.
14 The 5 HT 1A is to date the m ost extensively studied and characterized serotonin receptor, due to early identification of its binding site and a somewhat selective agonist, 8 OH DPAT. The 5 HT 1 receptors are coupled to the G i /G 0 family of G proteins, the response of activation to whi ch is the inhibition of a denylate cyclase. Brain 5HT 1 A receptors are located both pre and post synaptically. (Barnes and Sharp, 1999). Functions of 5 HT1 A and 5 HT1 B. The 5 HT 1A and 5HT 1B receptors have been implicated in numerous physiological and be havioral functions, most observed with the use of receptor agonists/antagonists or genetic modification in rats and mice. Unintended side effects of both experimental techniques remain a possibility, but several functional correlations have been well conne cted to 5 HT 1A and 5 HT 1B. Sleep wakefulness cycles have been well connected to 5 HT 1A with both genetic and pharmacological experiments. Mice genetically altered not to express 5 HT 1A or wild type mice given 5 HT 1A receptor antagonists typically exhibit paradoxical sleep wakefulness cycles as well as reduced REM periods (Boutrel, et al. 2002, Monti and Jantos, 2004). Both 5 HT 1A and 5 HT 1 B are implicated in regulation of anxiety behaviors. 5 HT1 A knockout mice show an increased tendency to avoid nov el and fearful environments and display anxiety like behaviors (Parks et al. 1998, Ramboz et al. 1998, and Heisler et al. 1998). Conversely, 5 HT 1 A mice modified to over express the 5 HT 1 A receptor protein during postnatal development were observed to ha ve elevated serotonin levels and displayed decreased anxiety like behavior (Kusserow, et al. 2004).
15 Pharmacological studies of 5 HT 1B show a similar relationship, with agonists producing an increase in anxiety and antagonists a decrease ( Chojnacka Wjcik, et al. 2005 and Tatarczynska et al. 2004). Both receptors also seem to influence learning and memory. Pharmacological studies on humans indicate that 5 HT 1A agonists are reported to have detrimental effects on human explicit memory; antagonists generally have an opposite effect (Buhot et al. 2000, Yasuno et al. 2003). Similar studies on rodents indicate that the stimulation of 5 HT 1A receptors generally produces learning impairments by interfering with memory encoding mechanisms. Antagonists of 5 HT 1A rec eptors conversely facilitate certain types of memory by enhancing hippocampal/cortical cholinergic and/or glutamatergic neurotransmission. (Ogren, et al 2008). Another study focusing on prefrontal cortex dependent learning and memory in knockout mice indi cate that 5 HT 1B receptors have an important role on impulsivity and a minor role in on prefrontal cortex dependent learning and memory, while 5 HT 1A receptors are implicated in facilitation of autoshaping, but their role in impulsivity and prefrontal cort ex dependent learning and memory appears to be limited (Pattij, et al. 2004). When contextual learning was studied using a passive avoidance task, mice given a 5 HT 1B agonist performed poorer than control studies, and mice given an antagonist or a combinat ion of the antagonist and agonist dose dependently improved performance, indicating that blockade of 5 HT 1B receptors facilitates this type of learning (Eriksson, et al. 2008).
16 Monoamine neurotransmitters such as serotonin act in the synaptic cleft, the gap between two nerve cells. The pre synaptic cell releases the neurotransmitter into the synaptic cleft, where it acts upon the receptor protein in the post synaptic cell. A small proportion of the neurotransmitter is lost d uring this process, but most is then re moved by monoamine transporters (specific for individual neurotransmitters) in the cell membrane of the pre synaptic cell, where it can then be released again, an overall process called release and reuptake. This term inates the action of the neurotransmitter within the cleft, as well as allowing it to be reused (Barker and Blakely, 1995). whose short term action is the selective, negativel y allosteric modulation of the pre synaptic serotonin transporter, preventing serotonin reuptake into the pre synaptic cell. The serotonin transporter is a molecular complex including an enzyme and protein with multiple binding sites. One site binds seroto nin itself, another binds to Na+, and a third binds to the SSRI. The Na+ binding site increases the transporter affinity for serotonin, while the SSRI binding site inhibits serotonin binding. When the transporter is inhibited by an SSRI, serotonin accumula tes in the synaptic cleft, increasing the concentration of serotonin and allowing it to act upon the post synaptic receptor again and again (Stahl, 1999). Serotonin, Behavior, and Teleosti. The specific actions of 5 HT receptors aside, synaptic seroton in concentration has been linked to a multitude of behaviors and clinical phenomena in humans, the most well
17 known of which is probably depression. In several studies, tryptophan and/or serotonin depletion has resulted in lowered mood states, sleep disorde rs, and other symptoms of clinical depression in normal patients and, and an increase in depressive symptoms in patients already exhibiting mild depression (Meltzer, 1990, Young et al. 2002). Other experiments have likewise linked serotonin to anxiety diso rders (Charney, 1990 and Yamauchi et al. 2006), eating disorders (Steiger, 2004), and stress (Simonenkov and Fedorov, 2002). As previously discussed, the serotonergic system in rats, mice and humans is also likely to be heavily involved in learning and m emory via the 5 HT 1A and 5 HT 1B receptors. Other experiments modifying concentrations of active serotonin have taken place. In rats, daily fluoxetine administration has been shown to impair two way active shuttle box avoidance learning in a manner that was not caused by sensory or motor impairment (Nelson, et al 1997). In another study, rats given chronic fluoxetine injections showed a marked detriment in hippocampal independent short delay appetitive Pavlovian conditioning tasks and an object recognition task in comparison to rats given saline injections, but neither group was significantly different in performance from a hippocampal dependent spatial water maze task (Valluzzi and Chan, 2007). Rationale for current experiment. In 2004, with a shuttle b ox avoidance task Fowler compared the response s of goldfish administered by WAY 100,63 5 a selective 5 HT 1A antagonist and fluxoetine The current experiment extends this research to measure the effects of increased dosage and the possible interaction of a 5 HT 1A antagonist and an SSRI, fluoxetine (Prozac).
18 WAY 100,635 WAY 100,635 was developed as a 5 HT 1A antagonist, and was thought at the time of the previous experiment to be selective for only this receptor. It ha d been used as the selective ant agonist of choice for the 1A receptor since its discovery in 1995. Since then, studies have implicated WAY 100,635 and its major human metabolite, WAY 100,634 as a dopamine D 4 full agonist, and a weak D 2L agonist, suggesting that the drug may not be as selective as originally thought. Unexpected pharmacological action of WAY observed in ongoing experiments prompted investigators to submit WAY to the NIMH sponsored Psychoactive Drug Screening Program. Cloned dopamine receptors and transporters in cell cultures were grown by this program, and subjected to radioligand binding assays. The results of these experiments suggest that WAY 100,635 is only tenfold more selective in its affinity for 5 HT 1A receptors over dopamine D 4 receptors, which, at concentrations commonly used in receptor studies, would lead to significant binding at both receptors rather than only at the serotonin receptor intended (Chemel, et al. 2006). However, a contrasting study indicates that WAY 100,635 may have a much higher selectivity for 5 HT 1 A receptors versus dopamine D 4 receptors, and supports the assertion that WAY 100,635 is a highly selective 5 HT 1A antagonist. This study also used radioligand binding, as well as functional assays on cell cultures. The results of these assays showed a hig h affinity for 5 HT 1A and low or moderate affinities for dopamine receptors, with the potency being more than 240 fold in favor of antagonism at 5 HT 1A than dopamine D 4. This study is more in line with previous experiments as to the selectivity of WAY as a receptor agonist/antagonist, and the authors suggest that the
19 apparent disparity in results between the two studies could be explained by the difference in cell lines used, with the former using human kidney cells that have a much higher level of tightl y coupled D 4 receptors, which may have accentuated the apparent affinity of WAY for those receptors (Martel, et al. 2007). Regardless, alternative antagonists were considered for the present study. Of known 5 HT 1A antagonists, few qualified for considerat ion as selective antagonists. WAY 100,135, a drug related to WAY 100,635, for example, is a 5 HT 1A antagonist, but also functions as a partial agonist for 5 HT 1B and 5 HT 1D (Davidson, et al. 1997). A drug called Lecozotan has been reported to be a selectiv e 5 HT 1A antagonist (Schechter, et al 2005), but as to date, there is little research into this compound, and most of what is alternative for WAY 100,635 was NAD 299. NAD 299 was formulated in 1997 and as yet is not in nearly as frequent use as WAY, but initial research indicates that it may be as good an antagonist for 5 HT 1A (J ohansson, et al 1997). However, the lack of easy availability of this compound, its lack of wa ter solubility (which would therefore require direct injection to administer to fish), and the comparative lack of related research using it in behavioral studies renders it inferior to WAY 100,635 for this experiment. It was therefore decided, given the e vidence supporting its selectivity, and the ease of administration, to retain WAY as the antagonist in the current experiment. This will also allow the current experiment to replicate exactly the previous experiment.
20 Fluoxetine Recent research has also shed some light on a probable explanation for the unexpected results of the fluoxetine group in the previous study. It is now apparent that fluoxetine may upregulate endogenus brain stores of allopregnanolone (Allo), and moreover that this mechanism occur s independently of 5 HT reuptake modification, and at doses too low to have a clinically significant 5 HT reuptake effect (Pinna, Costa and Guidotti, 2009). Allo is a neurosteroid that has a highly potent affinity to GABA A receptors, and acting as a posit ive allosteric modulator, and is the most abundant brain neurosteroid acting upon GABA A receptors (P ina et al. 2003). Allo has been shown in rats to improve deficits in passive avoidance retention (Romeo, et al 1994), alleviate induced conditions of stre ss, aggression and anxiety (Pinna, et al 2008), and improve spatial learning (He, et al. 2004). also suffered from a decrease in allo levels at the brain and cerebrospinal fluid N on psychiatric patients had an approximately two fold higher concentration of Allo, and treatment with fluoxetine subsequently normalized CSF Allo levels (Uzunova, et al. 1998). Later, it was shown that fluoxetine upregulates neurosteroid content at do ses incapable of inhibiting 5 HT reuptake. In dose depende nt studies, fluoxetine was seen to reverse the decrease of brain Allo levels induced by stress at levels that were ten to fifty times lower than the dosage required to significantly inhibit 5 HT re uptake. In these studies, corresponding improvements in behavioral deficits also occurred, even in experiments wherein 5 HT synthesis was inhibited pharmaceutically before the trials began (Pinna, Costa and Guidotti, 2009).
21 Interestingly, fluoxetine, whic h is a racemic mixture of two stereoisomers, S fluoxetine and R fluoxetine, displayed a ster e ospecific effect on neurosteroid biosynthesis. R fluoxetine, S fluoxetine, and R norfluoxetine (the major metabolite of R fluoxetine) all increased Allo biosynthes is at doses far lower than that required for 5 HT reuptake inhibition, while S norfluoxetine, (S affected Allo synthesis at levels fifty five times higher than that required for 5 HT reuptake inhibition (Pinna, et al 20 06). enough to ameliorate allopregnanolone deficits caused by stress without being high enough to have a significant effect on 5 HT accumulation in the brain, this could explain the lack of significant interaction between fluoxetine and control groups observed in this exp eriment. Unfortunately, there are still very few data available on dosages of fluoxetine required to produce serotonin reuptake inhibition in fish. One study indi cated that the 96 hr LC 50 dose for sheepshead minnows the same approximate size and weight as fish used in the previous study was less than 2.000 mg L 1 (in comparison, the dose given in the These data give considerable leeway to increase the dosage of fluoxetine while still keeping well within safe mortality limits (Winder, et al 2008). Using a still arbitrary, but substantially higher double dosage of fluoxetine in the current study is intended to address the dose dependency question, allowing fluoxetine to adequately function as a means of increasing brain serotonin levels.
22 Co Administration of WAY 100,635 and F luoxetine. Recently, there has been some research concerning co administration of WAY 100,635 and fluoxetine. Rats trained to perform a putative short term memory task were given WAY and fluoxetine separately and in tandem. WAY alone had no significant ef fect, while fluoxetine alone impaired response accuracy. Interestingly, when pre treated with WAY and then given fluoxetine, this deficit was not only reversed, but the rats showed a higher performance than either group. This suggests that fluoxetine impro ves short term memory function when 5 HT 1A receptors are blocked (Fernandez Perez, 2005). One explanation is that WAY 100,635 prevents desensitization of 5 HT 1A autoreceptors (Castro, et al 2008). As part of the current experiment, one group will be co administered WAY 100,635 and fluoxetine at the same dosages as the other groups so that the interaction of these two drugs and their affect on shuttle box avoidance testing can be studied. Materials and Methods. Although a perfect duplication of methods used by Fow l er (2004) was not possible due to budget, time constraints, random variability, and other factors beyond the experimenter's control, in general, all materials and methods used in this experiment match those from the prior experiment whenever p ractical, except for changes in experimental variables under study (such as Fluoxetine dosage, or the addition of the Fluoxetine WAY co administration group). Other changes not intended to be experimental variables will be compared and explained in this se ction in addition to a generalized explanation of materials and methods.
23 Subjects. Four groups of fifteen fish each (totaling sixty subjects) of species Carassius auratus auratus were obtained from local distributor Seascape Aquarium Supplies. The fis h were obtained via live capture from a native environment rather than captive breeding, in one group of 45 (for Fluoxetine, WAY, and control subject group) and one group of 15 (for Flu WAY co adminsitration group). Although in the previous (2004) experime nt all subjects obtained were of the comet variety, this was not possible in the present experiment due to availability limitations. Instead, approximately 5 10% of each group was of a different variety similar in size and weight. These individuals were as signed randomly across all groups using six sided dice. Subjects weighed an average of 7.5g and were 5.5 to 9.5cm in length (see appendix for individual subject length and weights), an average size close to those used in the 2004 experiment. After being brought to the laboratory, subjects were housed in a large (55 gallon) communal tank treated with antibiotic and anti parasitic medications for three days to allow time to acclimate to laboratory conditions. This was foreshortened from the nine days accli mation time in the previous experiment. T hree subjects from the first group died during acclimation due to unknown causes (most likely stress or age). At no point during the experiment were signs of significant or unusual parasitic, bacterial, or other inf ectious agents present in the subjects. Subjects were monitored in home tanks throughout the experiment, and in general, all groups showed consistent and similar levels of resting behavior, activity and food consumption, regardless of drug treatment.
24 H ome Aquaria Throughout the course of the experiment all subjects were housed in same aquaria used in the prior experiment. These consisted of fifteen 20 gallon (75 liter) aquaria measuring 32 cm x 76 cm x 32 cm. Each aquarium was divided into three parts with non transparent plexiglass dividers with evenly spaced holes approximately 5 cm apart. This allowed for easy identification of individual subjects as well as reducing subject interaction and the formation of dominance hierarchies while permitting che mo sensory communication between subjects to reduce the sensation of isolation. Although tanks had a larger capacity, for the current experiment they were filled only to 50 liters in order to increase the concentration of treatment drugs. As in the previ ous experiment, it was decided to forgo deionized tank water in favor of tap water due to concerns about maintaining proper solute concentration when moving subjects between home and experimental tanks. Chlorinated water was stored for a period of 24 hours before use in tanks to allow chlorine and other potentially harmful additives to evaporate out of solution. Oxygenation was provided by laboratory pumps filters were not u sed in the home tanks because of concerns that these may interfere with water soluble pharmaceutical treatment. Substrate in the form of symbiotic cultured bacteria was provided in the previous experiment due to concerns about harmful bacteriological conta mination amongst the subjects, but was deemed unnecessary in the current experiment. Instead, fecal matter and other tank detritus not absorbed by the foam filter was suctioned out via siphon during tank water change and replacement.
25 Both home and experi mental tanks were housed in the Pritzker Marine Biology Research Center Heating devices were not used in the tanks, and tank water temperature was maintained at an average of 22C through laboratory climate control, an appropriate mean temperature for the experimental subjects. Aquaria were arranged against the west wall of the laboratory on a rack with tanks four across and four down. Tanks on the bottom row were insulated against the concrete floor by foam plates. Large windows on the north wall allowed natural illumination during the day without subjecting the tanks to direct sunlight. Artificial illumination was provided by fluorescent room lights and only used when the experimenter was present. Access to this room of the laboratory was limited to the experimenter, marine lab personnel, and another thesis student conducting a concurrent project within the same facility. Maintenance and Feeding Feeding consisted of dropping a small pinch of flakes (1 2g) in each subject's tank section and observing to the rate at which it was consumed, then either providing additional food or proceeding to the next subject as warranted to prevent over feeding. During feeding, all subjects w ere observed closely for signs of stress or disease and their condition was noted. Tank water was changed once over the course of the thirteen day experiment, as described further in the pharmacological treatment section. Some evaporation of tank water wa s noticed. While additional water was added in the previous experiment to counter this, it was decided in the current experiment not to replace evaporated water due
26 to possible dilution of pharmaceutical concentrations. Over the course of the experiment, i t is estimated that less than 1.5 liters of water per tank was lost due to evaporation. A total of seven subjects died over the course of the experiment, two from the WAY group, one from the fluoxetine WAY co administration group, one from the fluoxetine group, and three from the control group. Given the apparently random distribution between groups and times of death, with the highest number of deaths found in the untreated control group at random intervals, it is unlikely that these deaths were caused b y experimental pharmaceuticals or overdose. Instead, likely factors include stress, natural lifespan, internal (non communicable) disease, or other unknown causes. After subject death, bodies were removed as soon as possible and disposed of to prevent fou ling of tank water. Shuttle Box Aquaria. As with the home tanks, the same shuttle boxes constructed by Dr. Beulig used in the previous experiment were used in the current experiment. Eight shuttle boxes (numbered 0 through 7) were available for use. Ho wever, after photo sensors in shuttle box #2 recorded unusually high numbers of escape events (in one case, 28 events in one trial) it was decided to reassign fish from this box and forgo using it to prevent possible data anomalies arising from oversensiti vity of the recording equipment. Since the first day of training has such a low impact on total avoids, it is unlikely that this reassignment had much of an effect on the data. Shuttle boxes were composed of black acrylic resin without interior markings All shuttle boxes were of uniform size, measuring 11.5 x 40.5 x16.5 cm. Inside each
27 shuttle box, a sloping partition divided each box into two sections of equal proportions. Water was added to a level approximately 2.5 cm above the peak of this divider allowing fish to cross back and forth between sides when motivating stimulus was present, but making it sufficiently difficult enough to prevent random crossings (see fig. 2). Drains set into the bottom of each box allowed the water to be changed between sessions. Each shuttle box was mounted within a wooden cabinet with sealing to prevent illumination from external sources. Two small lights ( 28 V) were mounted inside this cabinet, one over each side of the shuttle box, to provide the conditioned stimul us. Inside each shuttle box, stainless steel wire mesh electrodes mounted on either side of each section. These electrodes were connected to a Powerstat Variable Autotransformer, allowing creation of an low intensity pulsed AC electrical shocks that served as the unconditioned stimulus. An infrared photobeam atop the divider of each box was used to detect fish crossing the divider. The lights, photobeam, and shock stimulus were all connected to a HS Habitest Universal Linc, allowing PC control and recording of the entire experiment.
28 Fig. 2. Shuttle box with central hurdle, photoelectric beam, and stainless steel electrodes. This is supported on a retractable platform within the isolation chamber. Graphic State Notation software by Colburn Instruments was used in conjunction with a desktop computer running Microsoft Windows 95. This software interfaced with the Habitest Universal Lincs to provide control and recording of experimental events and data. Using the software, four states were programmed to be run during each experimental session: State 1: Lights off, Shock off. State one served as the initial trial condition. When entering state one, both the lights (conditioned stimulus) and the shock (uncondit ioned stimulus) were set to off. Transition into State 2 was set to occur randomly 52% of the time, checked every thirty seconds. This allowed for a variable inter trial interval to ensure responses are based upon the conditioned stimulus and not the passa ge of time.
29 Infrared photo detector triggering in State 1 was not recorded. After entering State 1 sixteen times, the program exited to Fin, allowing for fifteen trials per session. State 2: Lights on, Shock off. During state 2, the conditioned stimul us (lights) were turned on. Photo sensor triggering during State 2 was recorded as an Avoid, and was followed by immediate exit back into State 1. If the photo sensors were not triggered after ten seconds, the program exited to State 3. State 3: Lights o n, Shock on. Upon entry into state 3, the program engaged the variable auto transformer to send pulsed AC shocks through the electrodes at a 3.8 V intensity and 500ms duration once per second for five seconds. Triggering the photo sensors during State 3 ca used the program to exit to Stage 1 and record the event as an Escape. If the photo sensors were not triggered after five seconds, the program exited to State 4. State 4: Lights off, Shock off Upon entry into State 4, both stimulus were turned off. Aft er two seconds into State 4, the trial was recorded as a Failure and the program exited to State 1. Fin : After fifteen trials, the program ended and compiled the results of the session. Drug Administration WAY 100,635 and racemic Fluoxetine Hydrochlo ride were administered separately or in combination with three of the four experimental groups. Dosages and administration of these drugs differed significantly from the previous experiment in order to introduce the new experimental factors of the current study. As well, due to availability
30 and expense of the drugs used, tanks were filled to 50 L rather than 75 L in order to increase concentration by lowering the diluting effect of excess tank water. This change in water level was applied to all groups, inc luding control, in order to reduce its effect on experimental results. WAY 100,635 (N [2 [4 (2 Methoxyphenyl) 1 piperazinyl]ethyl] N 2 pyridinylcyclohexanecarboxamide maleate salt) was administered to the WAY experimental group as well as the WAY+Fluoxet ine experimental group. As previously noted, WAY is a selective 5 HT 1A receptor antagonist frequently used in research relating to effects and chemistry of serotonin. WAY administered to both groups was obtained from Sigma Aldrich Chemical. WAY has a molec ular mass of 422.56 g/mol, appears as a white crystalline solid, and has a solubility in water of 25 mg/ml. Fig 3: Chemical structure of WAY, 100,635. Image obtained from Sigma Aldrich Product Information reference. Racemic Fluoxetine Hydrochloride (LY 110,140 hydrochloride, () N Methyl [4 (trifluoromethyl)phenoxy]benzenepropanamine hydrochloride) was administered to the Fluoxetine as well as the WAY+Fluoxetine experimental groups. Fluoxetine is a selective serotonin reuptake inhibitor commonly sold under the trade name Prozac and is frequently prescribed as an antidepressant medication. Fluoxetine administered to both
31 groups was also obtained from the Sigma Aldrich Although some studies suggest that the S isomer may be more effective at inhibi ting the reuptake of serotonin without affecting production of related neurosteroids (Pinna, et al 2006) it was decided to use a racemic mixture in order to make dosage the primary experimental variable in the Fluoxetine group and to match the previous st udy. Fluoxetine is a white crystalline solid with a molecular mass of 345.8 g/mol and a solubility in water of 4 mg/ml. Fig 4: Chemical Structure of Fluoxetine Hydrochloride. Image obtained from Sigma Aldrich Product Information reference. As with the previous experiment, a variety of drug delivery methods were examined, such as direct injection, injection of fat soluble solutions, and treatment of tank water. This latter was eventually adopted for the present study, as it both matches the previous stu dy without introducing new experimental variables (such as the stress of daily injections) and remains the most practical solution for drug delivery of water
32 soluble compounds to fish. It was assumed that by direct treatment of aquarium water, the subjects would absorb medications in a dose dependent manner through chronic exposure throughout the trial, although this was not verified through tissue analysis or other means in the current experiment. In the current experiment, 5 mg of WAY 100,635 was disso lved in 50 ml of deionized H 2 O, and 8 ml of the resultant solution was added to each WAY group tank (filled with 50 L of water), resulting in a concentration of approximately 16 g/L matching the concentration used in the previous study. For the fluoxet ine group, 60 mg of Fluoxetine Hydrochloride was dissolved in 60 ml of deionized H 2 O, and 8 ml of the resultant solution was added to the appropriate tanks, resulting in a concentration of 160 g/L, approximately double the dose given in the previous study (81 g/L). In the WAY+Fluoxetine group, treatment procedures used for the WAY and fluoxetine groups were combined, resulting in a concentration of 160 g/L fluoxetine and 16 g/L WAY 100,635. The control group was given sham doses of deionized water in the same amounts. Across all groups, drug treatment was given the night before the first experimental session. Tank water was partially drained and replaced midway through the experiment, after session 7 and before session 8. No experimental trials took place the day tank water was changed. In all groups including controls, the amount of water removed during the tank change and lost through evaporation was measured and replaced to keep tank water level at 50 L, and appropriate amounts of WAY, Fluoxetine, or sham solutions were added to keep drug treatments at the appropriate level.
33 Subject Assignment, deaths, and anomalies. Each subject was given an i ndividual label identifying it by group and number. For example, WAY10 indicates subject 10 of the WAY group, while subject FW12 indicates subject 12 of the Fluoxetine+WAY group. It was decided not to incorporate shuttle box ID, tank number, or tank partition into subject assignments as was done in the previous experiment due to the confusing nature of thi s type of subject identification when reviewing the study from raw data. Instead, each subject was assigned a shuttle box number randomly via rolls of a six sided dice until all boxes were filled, and then each subject was placed in a tank section correspo nding to the shuttle box in numerical order. For example, subject WAY1 and WAY7 were picked at random using the dice, then placed in tank 1, section 1, and tank 3, section 1, respectively, and both assigned to shuttle box 0. As was mentioned previously, sh uttle box 2 was shown to be malfunctioning after the first session, and these subjects were reassigned to random boxes. (For a list of subjects paired to shuttle box and tank placement, see appendix). Several subjects died during the course of the experi ment. CTRL14 died the night after administration of the sham drug treatment, before the first session the following day, after which FW15 died. CTRL4 was found dead upon beginning the third session. WAY2 and FLU4 were found dead on the day of the seventh s ession, and CTRL5 died the following day, before the eighth session. WAY11 jumped out of the shuttle box during the same session, but was revived after replacement into home tank. Finally, WAY9 died before the 10 th session. Following subject deaths, the re mains were removed from the
34 home tanks and disposed of with laboratory facilities, and incomplete experimental data from dead subjects was expunged from records used for statistical and data analysis. Experimental Protocol Experimental protocol for each session consisted of equipment testing, subject testing, equipment cleaning, and data collation. Thirteen sessions, one per day, with four subject groups were run, and each session consisted of fifteen trials. A concerted effort was made to begin each ses sion at the same time of day, and in general, experimentation began at approximately 12:30 PM and ended at 3:30 4:00 PM. Before each session, all shuttle boxes were tested by filling with water and engaging a test program in G raphic S tate. This program a llowed the experimenter to use a voltmeter to test each box's electrodes for proper conductivity, as well as test the infrared beam and lights to ensure that each box was functioning properly before use in the experiment. Prior to the initial session, tw o large (250 gallon) drums were half filled with tap water and allowed to dechlorinate though evaporation. These containers were then connected via a PCV pipe with closeable valve functioning as a siphon. A charcoal filter was connected to one drum and all owed to run continuously throughout the experiment, including in between sessions. By closing the siphon valve, it was possible to separate clean, filtered water from water used in the previous sessions, which was dumped into the container with the filter. After filtering for 24 hours, the siphon was opened, allowing the cleaned waste water to be reused for the day's sessions. This was done to help eliminate
35 traces of drugs excreted by each fish as well as fear pheromones and other hormones that would other wise be present in re used unfiltered water. Following filling and testing of each shuttle box, subjects were transferred from home aquaria into the boxes via net. At no point during the experiment were subject directly handled by the experimenter. Each shuttle box cabinet was then closed, (ensuring darkness during the trial aside from the conditioned stimulus light) and the experimental session was initiated and recorded via desktop computer. Sessions lasted approximately 13 to 23 minutes, depending upon randomly generated inter trial interval and subject performance. Sessions were monitored continuously during experimentation. At the conclusion of each session, subject was carefully removed with net and replaced in its appropriate tank slot. The shuttle box was then drained and refilled for the next subject. boxes were drained and wiped with clean paper towels to discourage the growth of mildew or other harmful organisms and subjects were fed as d escribed previously. Session data for each subject was then compiled by computer, averaged, and recorded. Results. Avoidance Responses. Thirteen sessions were recorded for all four groups. Sessions were further subdivided into three blocks of time, with b lock one comprising the first five sessions, and blocks two and three comprising four sessions each consecutively. Figure 5 shows the result of mean Avoid responses graphed over each session number. As can be seen, there was a high degree of variability i n performance across all groups, but in general, the WAY group outperformed other groups in all but one session,
36 while the WAY+FLU group consistently underperformed in comparison to all other groups. Total responses over all sessions for each subject are a lso included in Table 2 to provide a numerical overview of resulting data. This table also helps to illustrate high degree of variability in individual responses with some subjects performing very well and some responding very poorly, even within groups. Fig 5: Mean avoids of each group plotted across all sessions.
37 excluded from calculations due to mid trial death. Total of Responses over all sessions. Subject Avoids Subject Avoids Subject Avoids Subject Avoids W1 58 C1 118 F1 3 FW1 0 W2 X C2 8 F2 5 FW2 61 W3 94 C3 18 F3 36 FW3 13 W4 21 C4 X F4 X FW4 0 W5 21 C5 X F5 7 FW5 0 W6 6 C6 40 F6 24 FW6 42 W7 73 C7 1 F7 14 FW7 25 W8 18 C8 3 F8 2 FW8 0 W9 X C9 1 7 F9 105 FW9 13 W10 28 C10 14 F10 0 FW10 6 W11 67 C11 3 F11 1 FW11 1 W12 13 C12 4 F12 2 FW12 6 W13 1 C13 16 F13 2 FW13 18 W14 0 C14 17 F14 29 FW14 14 W15 41 C15 X F15 21 FW15 X Mean 33.92 Mean 21.58 Mean 17.92 Mean 14.21 Table 3 summarizes descri ptive statistical values of the total avoids of subjects in all treatment groups over all sessions. The WAY group had the highest mean Avoids of all groups at 33.92, while the WAY and Fluoxetine combination group had less than half this value at 14.21, fol lowed by the Fluoxetine group at 17.92 and the control group scored 21.58 mean avoids. The WAY group was also the only group to have a standard deviation of this value less than the value of the mean (30.18). The standard deviation of all other groups exce eded the mean, with the standard deviation of the control group being almost one third higher than the mean (32.17). At least one subject in every group except the control group failed to score any Avoids throughout the entire experiment. Additionally, the control group featured the individual with the highest total number of Avoids. The WAY and Fluoxetine combination group scored both the lowest maximum number of Avoids as well as the lowest mean, and second lowest median.
38 Table 3: Descriptive Statistics of total avoidance responses per group over all sessions. WAY Fluoxetine WAY and Fluoxetine Control Mean 33.92 17.92 14.21 21.58 Standard Deviation 30.18 27.69 18 32.17 Min 0 0 0 1 Median 21 6 9.5 15 Max 94 105 61 118 N 13 14 14 12 A two way ANO VA of avoid frequencies of treatments over blocks was hoc test was performed to further analyze the results. Overall analysis of both blocks and treatments show statistical significance; F value for blocks was 3.1 4 with a P value of .0382, while treatment F value was 3.08 and P value was .0295. Averages consistently improved across blocks, with WAY showing the highest improvement. Table 4 displays mean avoid responses between groups showing significant differences at the .05 level across blocks of time. No group was found to be significantly different from the control, which maintained a fairly median level of performance, but the WAY and WAY/Fluoxetine combination groups were found to be statistically different. A dditionally, performance across block 1 was found to be statistically different from block three, indicating statistically significant improvement in avoidance learning over time, however, block two was not significantly different from blocks 1 and 3. Tab same letters are significantly different at the .05 value. Mean Avoids WAY Fluoxetine WAY and Fluoxetine Control Block 1 7.00 A,I,C 4.35 B 1.93 C, I,K 4.42 D Block 2 12.62 E,K,G 6.21 F 4.93 G,A 8.33 H Block 3 14.30 A,J,C 7.35 B 7.36 C,J,K 8.83 D All Blocks 33.92 L 17.92 M 14.21 L 21.58 N
39 Figure 6 provides a comparison graph of mean Avoid responses by block and treatment, with a 95% confidence interval. Figure 6: Mea n avoids of treatment groups by block, with 95% confidence interval. Failures. Failure results were analyzed in a similar manner to Avoids to provide a basis of comparison in terms of negative performance. Figure 7 provides a graphical plot of the mean failures of each group for each session. Results in this category also show a high degree of variability, especially within the control group. As a result of this, it is difficult to generalize results, although the control group had the highest average fa ilure rate and
40 the fluoxetine group had the lowest failure rate with the failure rate somewhere between the two most of the time. Fig 7: Mean Failures of each group plotted across all sessions Table 5 provides an overview of i ndividual subject Failure responses totaled for all sessions. Once again it can be seen that some subjects within each group had very differing totals compared to other subjects within the same group.
41 Table 5: Total of Failure responses per subject over excluded from calculations due to mid trial death. Total of Failure Responses over all sessions. Subject Failures Subject Failures Subject Failures Subject Failures W1 20 C1 1 F1 31 FW1 137 W2 X C2 59 F2 60 FW2 2 0 W3 20 C3 11 F3 13 FW3 10 W4 21 C4 X F4 X FW4 35 W5 13 C5 X F5 43 FW5 149 W6 123 C6 2 F6 59 FW6 1 W7 7 C7 66 F7 55 FW7 46 W8 3 C8 95 F8 10 FW8 17 W9 X C9 55 F9 0 FW9 17 W10 31 C10 113 F10 35 FW10 1 W11 19 C11 82 F11 6 FW11 119 W12 130 C12 112 F1 2 66 FW12 126 W13 142 C13 35 F13 93 FW13 1 W14 114 C14 31 F14 21 FW14 13 W15 32 C15 X F15 11 FW15 X Mean 51.92 Mean 55.16 Mean 35.92 Mean 49.42 Table 6 provides descriptive statistics of Failure responses totaled for groups over all sessions. Of note is the low mean of the Fluoxetine group (35.92) relative to other groups (ranging from 49.42 to 53.5). All groups had a high standard deviation, with Control group having the highest range between standard deviation and mean (38.7 std with mean 53.5). The Way and Fluoxetine combination group was notable in that it had the highest maximum value (149) and the lowest median (18.5). Table 6: Descriptive Statistics of total Failure responses per group over all sessions. WAY Fluoxetine WAY and Fluoxetine Con trol Mean 51.92 35.92 49.42 53.5 Standard Deviation 53.19 27.65 56.42 38.7 Min 3 0 1 1 Median 21 33 18.5 57 Max 142 93 149 113 N 13 14 14 12
42 To determine if negative performance was significant between groups and over blocks of time, a two way ANOV hoc test was also performed to determine statistical significance between individual groups and blocks. Statistical significance was not found overall, nor between blocks or between groups. Analysis of signi ficance between treatment groups indicated an F value of 1.22 and a P value of .3056, while analysis between blocks of time indicated an F hoc test confirmed these results, returning no treatment group or block as statistical ly significant from any other. Fig 8: Mean Fail responses by treatment type grouped by block, with 95% confidence interval.
43 Escapes Because of the derivative nature of escape responses (in that all responses not recorded as Avoids or Failures must be Escapes), these responses were not subjected to the same level of statistical analysis of Avoid and Failure responses. However, totals for each subject are provided in Table 6, and descriptive statistics are provided in Table 7 f or comparison. The fluoxetine group scored highest in escape responses overall with a mean response of 141.57, indicating a high level of activity, followed by the combination group. Table 6: dicate subject data excluded from calculations due to mid trial death. Subject Escapes Subject Escapes Subject Escapes Subject Escapes W1 132 C1 76 F1 161 FW1 58 W2 X C2 119 F2 130 FW2 114 W3 91 C3 165 F3 146 FW3 172 W4 153 C4 X F4 X FW4 160 W5 161 C 5 X F5 145 FW5 46 W6 70 C6 146 F6 112 FW6 152 W7 115 C7 128 F7 126 FW7 124 W8 174 C8 98 F8 183 FW8 178 W9 X C9 123 F9 90 FW9 165 W10 121 C10 68 F10 160 FW10 188 W11 109 C11 110 F11 188 FW11 75 W12 52 C12 94 F12 127 FW12 63 W13 47 C13 144 F13 100 FW 13 176 W14 79 C14 147 F14 148 FW14 168 W15 110 C15 X F15 166 FW15 X Mean 108.7692 Mean 118.16667 Mean 141.571 Mean 131.3571 Table 7: Descriptive Statistics of total escape responses per group over all sessions. WAY Fluoxetine WAY and Fluoxetine Cont rol Mean 108.76 141.57 131.35 118.16 Standard Deviation 40.22 28.23 50.85 30.06 Min 47 90 46.00 68 Median Max 188 188 188 165 N 13 14 14 12
44 Discussion. Unfortunately, the results of the experiment do not provide clear statistically significant differences between the treatment groups and the untreated control group. Most likely, this is due to the high variability between individual subjects within each group. However, support for avoidance learning for all groups is strong, given the statistic ally significant difference between avoid responses performed during block one and block three. Additionally, there is a clear statistically significant difference between the WAY and fluoxetine / WAY combination group, suggesting a possible interaction ef fect when fluoxetine is combined with WAY 100,635 resulting in a decrease in performance in avoidance tasks. Finally, by comparing overall performance trends between treatment groups to those of the prior experiment, conclusions can be drawn about the effe cts of each treatment. Performance Variability. Currently, no data are available to quantify the cause of the high levels of performance variability within groups. In general, most subjects who performed poorly did so consistently. For example, all group s except the control group had at least one subject which failed to record a single Avoid response throughout all thirteen sessions. This suggest that variance in subject performance may be the result of contributing factors with influences stronger than t hose of the treatments given. Several factors could be responsible in part or whole for influencing individual performance variability. The overall cognitive capabilities between individual subjects was probably a strong contributor, as avoidance learnin g is dependent upon several cognitive and behavioral
45 factors such as memory, base anxiety and stress levels which tend to vary somewhat among individuals. Environment may have contributed to high subject response variability. Although all efforts were ma de to keep subjects in a consistent environment with little influence over subject stress levels and behavior, some environmental factors were outside the control of the examiner. However, since the environment in which subjects were housed was identical i n location and equipment to previous studies which did not suffer from variability issues (see Fowler, 2004) it is unlikely that environmental effects were significant. Testing equipment may also have influenced variance in results between individuals. E fforts were made to keep shuttle boxes clean and well maintained, and shuttle boxes were tested with a volt meter prior to every session (as described previously, pg. 35 and 36). However, at the time of the experiment, shuttle box and related computer appa ratus used were several years old and may have deteriorated somewhat in performance over time. One shuttle box was found to be grossly oversensitive and was not used in the experiment. Others may have suffered from less obvious issues with sensitivity of i nfrared recording or variances over the course of a given session in voltage output of the unconditioned stimulus. Subjects tested in shuttle box #5 in particular were noted to have consistently poor performance, although testing could not produce any reli able defect in shuttle box operation, and by the time the effect was noticed, the experiment was well underway and transferring subjects to a different box would have been impractical and ill advised.
46 Some factors can probably be eliminated as possible in fluences of subject variance. Drug quality and administration was most likely consistent for all subjects, as drugs administered were obtained from a reliable and respected supplier and were therefore unlikely to include significant contaminants. The metho d used for drug administration was also unlikely to be at fault, since subjects housed within different sections of the same aquaria had no consistently positive or negative trends in performance. Individual metabolism and body mass may have had a minor ef fect on relative rates of absorption of treatment drugs from aquaria water, but as subjects were randomly distributed and variation of mass and length of subjects was minor, this is unlikely to be a factor. Additionally, the control group, which was not gi ven a drug treatment, also showed very high performance variability, as can be seen from Tables 2 and 5. Although several subjects died over the course of the experiment, external biological influences such as disease or parasite infestations are unlikely to have contributed to response variability, as subject deaths occurred in separate home tanks without spreading to other members of the same tank, and did not appear to be the result of a communicable infestation. Additionally, subjects that died over the course of the experiment did not display sickly behavior before death when inspected visually. Comparisons between groups. There is strong statistical evidence that the experiment was a success in teaching the avoidance task to members of all subject g roups. Each group averaged a higher number of avoids over time, as shown by figures five and six. Additionally, there was a statistically significant difference between performance of all groups between blocks one
47 and three (see table 4), indicating a real improvement over time. Block two was not different enough from the other two blocks to register as statistically significant, suggesting a rate of improvement over time that is moderate, rather than drastic. Failure rates also dropped overall in all group s, although the progression over time was not as clear (see Fig. 8 ) and the failure results between blocks of time were not statistically significant. Overall, this result is important to the experiment as a whole, indicating that despite high variability, the overall goal and means of testing performance between groups was valid. In general, the WAY group outperformed the other groups, showing the highest average increase in avoid responses, followed by the control group, while the WAY and Fluoxetine+WAY groups ended approximately tied. During the last block of time in the trials, fish given WAY 100,635 were performing nearly twice the number of avoids on average as fish in other groups. Finally,in addition to learning the task more reliably overall, WAY f ish learned much faster, outperforming all other groups by a comparatively large amount even during the initial block. The control group, as might be expected, had a median level of performance in comparison to other groups, nearly matching the fluoxetine group in mean avoids during the initial block, and pulling slightly ahead during the final two blocks. This result is also encouraging, as although the differences were not statistically significant, a baseline for general comparison can still be establis hed. The fluoxetine group was the second lowest in mean avoids overall, with some interesting trends over time. During the first block, this group was just under the control group (by less than one average avoid), a result which is consistent with studies
48 indicating that fluoxetine may have an initial delay between administration and onset of action (Thompson, 2002). Likewise, during the last block, average fluoxetine performance was almost equal to the combination group, suggesting some validity to the id ea that co administration of fluoxetine with WAY 100,635 may result in a reduction of delay between treatment and onset of action (Hjorth et al. 2000). Finally, the fluoxetine and WAY co administration group had the fewest average avoids overall. Addition ally, this group performed noticeably fewer avoids than other groups during the first two blocks, indicating a swift mechanism much like the WAY group. This group was also low enough in performance in comparison to the WAY group to achieve a statistically significant difference despite the high variability of results, indicating a strongly negative effect occurring when fluoxetine is administered in combination with WAY compared to administration of WAY alone. Present experimental data is not enough to dete rmine if this effect is an amplification of the suspected negative performance effects of fluoxetine alone, an effect that reduces the time dependent factor in fluoxetine treatment, or a novel effect entirely resulting from interactions between the drug gr oups and the 5 HT system. Regardless, the door has been opened for further examination of this surprising result and possible mechanisms of action. Support of Hypotheses and Conclusions. As in the previous experiment, the current experiment did not sh ow significant difference s between control and other treatment groups. Regardless, an examination of the overall performance trends in the two experiments provides some illuminating comparisons.
49 Overall, the trends of the data from the current experiment are consistent with those from the previous experiment. In both experiments, the WAY group outperformed the other groups in every block. In this respect, the experiment successfully verifies the results of the previous experiment providing even more supp ort for the hypothesis that 5 HT 1A antagonists improve avoidance task performance in fish and easing potential doubt about WAY's potential action as a minor D 4 receptor agonist. Likewise, the fluoxetine group in both experiments came in under, but not si gnificantly different, from the control. The high level of data variability may have swamped treatment effects in the present experiment. There is some indication that the fluoxetine treatment did have an effect, as groups in both experiment s had average a void results mirroring the control group during the first block, suggesting that a delay between treatment and onset of action was present in both experiments, an effect typical for fluoxetine treatment. Despite this positive indication, it cannot be fai rly said that the current experiment provided support for the hypothesis that the previous experiment's lack of difference between controls and fluoxetine was due to fluoxetine levels too low to raise 5 HT levels which then failed to impair avoidance perfo rmance in the mechanism suggested by Pinna, et al. (2008). However, by the same reasoning, the current experiment also provides no data which precludes this possibility. As fluoxetine concentration was doubled, but apparently had little significant chance in relative performance of controls and fluoxetine groups, perhaps increasing the dosage even more may have provided a greater difference. It may well be worth once again repeating this portion of the experiment in future studies, with another increase in dosage and measures taken to reduce variability of responses.
50 Based upon a study by Fernandez Perez (2005) in which rats were co administered fluoxetine and WAY and given memory intensive tasks to perform, it was hypothesized that the fluoxetine and WAY co treatment group would see an increase in performance relative to control. Instead, this group performed so negatively in comparison to the WAY group as to provide the only significant treatment effect, as discussed previously. This result suggests inst ead that the opposite is true, and that WAY and fluoxetine co administration has a powerfully detrimental effect on performa nce tasks in fish. Presently, relatively little literature is available on 5 HT receptor antagonist and SSRI co administration in ec totherms which makes it difficult to speculate on why this is so. However, studies on the clinical effect on this kind of treatment combination are becoming more frequent. For example, a new drug (WAY 211,612) is currently being evaluated and has been fou nd to act as both a selective serotonin reuptake inhibitor and a selective 5 HT receptor antagonist (Beyer, et al. 2009). As this and other SSRI / 5 HT antagonist combination studies continue, a plausible explanation for this result may soon present itself and warrant future testing. Overall, the current experiment has proven fruitful, yielding good data on the performance of fish in avoidance learning when treated with these drugs, such as conf irmation of the beneficial effects of treatment with a recept or antagonist, a continuation of the trend of sub average performance and delay in effect of fluoxetine treatment, and a first glimpse into the unusual effects of co administration of these drugs in ectotherms. Several future experiments related to the c urrent one may be worth pursuing Running a group in which initial avoidance training period is conducted before
51 administration of treatments could help separate out cognitive memory and learning effects from stress and other behavioral effects. Studies us ing groups with variable fluoxetine dosage with staggered administration dates could provide additional clarity to dosage and delay effects. Investigations using other species of fish, or other ectotherms such as reptiles and amphibians, may help consolida te data across an evolutionary time line Further investigations into the unexpected results of the co administration group is certainly warranted, with studies in dose, mechanism, and cause all having the potential to yield novel information.
52 Appendix A: Raw Data. The following includes all raw data from the experiment in order of session. WAY and Fluoxetine: SESSION 1 ID Avoids Escapes Failures ID Avoids Escapes Failures W1 0 15 15 F1 0 15 0 W2 1 10 4 F2 0 11 4 W3 0 13 2 F3 3 4 8 W4 0 13 2 F4 0 15 0 W5 0 2 13 F5 0 15 0 W6 0 14 1 F6 0 1 14 W7 0 15 0 F7 0 10 5 W8 0 14 1 F8 0 13 2 W9 0 15 0 F9 1 14 0 W10 0 3 12 F10 0 15 0 W11 0 9 6 F11 0 14 1 W12 0 14 1 F12 0 8 7 W13 0 5 5 F13 0 0 15 W14 0 1 14 F14 0 15 0 W15 0 1 4 F15 0 15 0 TOTAL: 1 144 80 TOTAL: 4 165 56 SESSION 2 ID Avoids Escapes Failures ID Avoids Escapes Failures W1 0 15 0 F1 0 2 13 W2 0 11 4 F2 0 6 9 W3 1 10 4 F3 3 12 0 W4 0 1 14 F4 0 15 0 W5 1 14 0 F5 0 15 0 W 6 0 14 1 F6 0 11 4 W7 2 13 0 F7 0 4 11 W8 0 15 0 F8 0 12 3 W9 1 14 0 F9 2 13 0 W10 0 0 0 F10 0 15 0 W11 0 10 5 F11 0 14 1 W12 4 11 0 F12 0 12 3 W13 0 11 4 F13 0 0 15 W14 0 13 2 F14 0 15 3 W15 6 9 0 F15 0 15 3 TOTAL: 15 161 34 TOTAL: 5 161 65
53 SESSION 3 ID Avoids Escapes Failures ID Avoids Escapes Failures W1 0 15 0 F1 0 15 0 W2 9 5 1 F2 0 11 4 W3 15 0 0 F3 0 13 2 W4 0 13 2 F4 2 13 0 W5 0 15 0 F5 0 5 10 W6 0 5 13 F6 0 14 1 W7 4 11 0 F7 1 13 1 W8 0 15 0 F8 0 13 2 W9 0 15 0 F9 3 12 0 W10 0 4 11 F10 0 15 0 W11 0 12 3 F11 0 15 0 W12 8 7 0 F12 0 1 14 W13 0 0 15 F13 0 14 1 W14 0 8 7 F14 5 10 0 W15 5 8 2 F15 0 15 0 TOTAL: 41 133 54 TOTAL: 11 179 35 SESSION 4 ID Avoids Escape s Failures ID Avoids Escapes Failures W1 2 13 0 F1 2 9 4 W2 1 11 2 F2 1 11 3 W3 2 11 2 F3 0 15 0 W4 0 14 1 F4 6 8 1 W5 0 15 0 F5 0 5 10 W6 0 6 9 F6 2 13 0 W7 5 10 0 F7 0 15 0 W8 0 14 1 F8 0 14 1 W9 0 15 0 F9 13 2 0 W10 2 11 2 F10 0 14 1 W11 1 14 0 F11 0 15 0 W12 0 2 13 F12 0 1 14 W13 0 0 15 F13 0 15 0 W14 0 5 10 F14 2 13 0 W15 0 13 0 F15 0 8 7 TOTAL: 13 154 55 TOTAL: 26 158 41 SESSION 5 ID Avoids Escapes Failures ID Avoids Escapes Failures W1 6 9 0 F1 0 15 0 W 2 1 7 7 F2 0 13 2 W3 7 8 0 F3 0 15 0 W4 0 14 1 F4 1 14 0 W5 4 11 0 F5 0 6 9 W6 0 4 11 F6 1 13 1 W7 5 10 0 F7 1 12 2 W8 0 14 1 F8 0 15 0 W9 1 14 0 F9 14 1 0
54 W10 5 10 0 F10 0 14 1 W11 1 14 0 F11 0 15 0 W12 0 7 8 F12 0 6 9 W13 0 0 15 F1 3 1 12 2 W14 0 13 2 F14 5 10 0 W15 5 8 2 F15 1 14 0 TOTAL: 35 143 47 TOTAL: 24 175 26 SESSION 6 ID Avoids Escapes Failures ID Avoids Escapes Failures W1 1 9 5 F1 0 15 0 W2 0 2 13 F2 0 14 1 W3 14 1 0 F3 0 14 1 W4 1 14 0 F4 0 6 9 W5 2 13 0 F5 0 4 11 W6 0 4 11 F6 2 13 0 W7 6 9 0 F7 7 8 0 W8 2 13 0 F8 0 15 0 W9 1 14 0 F9 12 3 0 W10 5 10 0 F10 0 12 3 W11 5 10 0 F11 0 15 0 W12 0 1 14 F12 0 2 13 W13 0 0 15 F13 1 10 4 W14 0 7 8 F14 6 9 0 W15 12 3 0 F15 0 15 0 TOTAL: 49 110 66 TOTAL: 28 155 42 SESSION 7 ID Avoids Escapes Failures ID Avoids Escapes Failures W1 3 12 0 F1 0 14 1 W2 X X X F2 4 10 1 W3 10 5 0 F3 0 14 1 W4 0 14 1 F4 X X X W5 1 14 0 F5 2 12 1 W6 0 5 10 F6 0 4 11 W7 9 6 0 F7 1 14 0 W8 0 15 0 F8 0 15 0 W9 1 10 4 F9 1 14 0 W10 1 14 0 F10 0 15 W11 7 8 0 F11 0 15 0 W12 0 4 11 F12 0 13 2 W13 0 0 15 F13 0 3 12 W14 0 8 6 F14 2 13 0 W15 8 5 2 F15 3 12 0 TOTAL: 40 120 49 TOTAL: 13 168 29 SESSION 8 ID Avoids Escapes Failures ID Avoids Escapes Failures W1 5 10 0 F1 0 15 0 W2 X X X F2 0 10 5 W3 12 2 1 F3 2 13 0
55 W4 0 15 0 F4 X X X W5 2 13 0 F5 0 15 0 W6 0 3 12 F6 0 3 12 W7 10 5 0 F7 1 14 0 W8 1 14 0 F8 1 14 0 W9 1 8 6 F9 8 7 0 W10 7 8 0 F10 0 6 9 W11 6 4 5 F11 1 14 0 W12 0 1 14 F12 0 13 2 W13 0 0 15 F13 0 1 14 W14 0 5 10 F14 2 13 0 W15 3 9 3 F15 6 9 0 TOTAL: 47 97 66 TOTAL: 21 147 42 SESSION 9 ID Avoids Escapes Failures ID Avoids Escapes Failures W1 7 8 0 F1 0 15 0 W2 X X X F2 0 14 1 W3 3 10 2 F3 2 13 0 W4 2 13 0 F4 X X X W5 1 14 0 F5 3 11 1 W6 0 2 13 F6 0 2 13 W7 5 10 0 F7 0 15 0 W8 2 13 0 F8 0 15 0 W9 0 3 12 F9 10 5 0 W10 2 12 1 F10 0 14 1 W11 8 7 0 F11 0 15 0 W12 0 2 13 F12 0 15 0 W13 0 0 15 F13 0 1 14 W14 0 9 6 F14 3 12 0 W15 1 13 1 F15 7 8 0 TOTAL: 31 116 63 TOTAL: 25 155 30 SESSION 10 ID Avoids Escapes Failures ID Avoids Escapes Failures W1 10 5 0 F1 1 14 0 W2 X X X F2 0 10 5 W3 8 7 0 F3 9 6 0 W4 2 13 0 F4 X X X W5 6 9 0 F5 1 14 0 W6 0 3 12 F6 2 13 0 W7 5 10 0 F7 0 6 9 W8 0 15 0 F8 1 14 0 W9 X X X F9 12 3 0 W10 0 10 5 F10 0 14 1 W11 11 4 0 F11 0 15 0 W12 0 1 14 F12 0 14 1 W13 0 4 11 F13 0 11 4 W14 0 2 13 F14 0 9 6 W1 5 0 8 7 F15 1 14 0
56 TOTAL: 42 91 62 TOTAL: 27 157 26 SESSION 11 ID Avoids Escapes Failures ID Avoids Escapes Failures W1 8 7 0 F1 0 14 1 W2 X X X F2 0 8 7 W3 7 14 4 F3 7 8 0 W4 1 14 0 F4 X X X W5 3 12 0 F5 0 14 1 W6 0 2 14 F6 3 10 2 W7 12 3 0 F7 0 1 14 W8 5 10 0 F8 0 15 0 W9 X X X F9 12 3 0 W10 2 13 0 F10 0 11 4 W11 13 2 0 F11 0 13 2 W12 0 1 14 F12 0 14 1 W13 0 9 6 F13 0 12 3 W14 0 0 15 F14 0 10 5 W15 0 10 5 F15 0 14 1 TOTAL: 51 97 58 TOTAL: 22 147 41 SESSION 12 ID Avoids Escapes Failures ID Avoids Escapes Failures W1 8 7 0 F1 0 15 0 W2 X X X F2 0 10 5 W3 7 5 3 F3 5 9 1 W4 1 14 0 F4 X X X W5 1 14 0 F5 0 15 0 W6 0 1 14 F6 3 11 1 W7 10 5 0 F7 0 2 13 W8 7 8 0 F8 0 15 0 W9 X X X F9 11 4 0 W10 1 14 0 F10 0 15 0 W11 9 6 0 F11 0 13 2 W12 0 0 15 F12 1 14 0 W13 0 5 10 F13 0 9 6 W14 0 0 15 F14 0 9 6 W15 0 10 5 F15 2 13 0 TOTAL: 44 89 62 TOTAL: 22 154 34 SESSION 13 ID Avoids Escapes Failures I D Avoids Escapes Failures W1 8 7 0 F1 0 3 12 W2 X X X F2 0 2 13 W3 8 5 2 F3 5 10 0 W4 14 1 0 F4 X X X W5 0 15 0 F5 1 14 0 W6 6 7 2 F6 11 4 0 W7 0 8 7 F7 3 12 0 W8 1 14 0 F8 0 13 2
57 W9 X X X F9 6 9 0 W10 3 12 0 F10 0 0 15 W11 6 9 0 F11 0 15 0 W12 1 1 13 F12 1 14 0 W13 1 13 1 F13 0 12 3 W14 0 8 6 F14 4 10 1 W15 1 13 1 F15 1 14 0 TOTAL: 49 113 32 TOTAL: 32 132 46 Control and Combination Group: SESSION 1 ID Avoids Escapes Failures ID Avoids Escapes Failures C1 0 15 0 FW1 0 3 12 C2 0 1 4 FW2 0 0 15 C3 0 13 2 FW3 0 11 4 C4 0 5 10 FW4 0 11 4 C5 0 11 4 FW5 0 0 15 C6 0 15 0 FW6 0 12 3 C7 0 13 2 FW7 0 12 3 C8 0 10 6 FW8 0 15 0 C9 0 15 0 FW9 0 0 15 C10 0 11 4 FW10 0 15 0 C11 0 15 0 FW11 0 9 6 C12 0 11 4 FW12 0 3 12 C13 0 14 1 FW13 0 14 1 C14 0 14 1 FW14 0 15 0 C15 X X X FW15 X X X TOTAL: 0 163 38 TOTAL: 0 120 90 SESSION 2 ID Avoids Escapes Failures ID Avoids Escapes Failures C1 0 15 0 FW1 0 1 14 C2 0 12 3 FW2 0 13 2 C3 1 14 0 FW3 0 13 2 C4 0 8 7 FW4 0 11 4 C5 1 13 1 FW5 0 1 14 C6 0 14 1 FW6 0 14 1 C7 0 11 4 FW7 0 15 0 C8 0 9 6 FW8 0 15 0 C9 0 14 1 FW9 0 14 1 C10 0 2 13 FW10 0 15 0 C11 0 15 0 FW11 0 2 13 C12 0 15 0 FW12 0 3 12
58 C13 0 8 7 FW13 0 15 0 C14 0 1 14 FW14 0 14 1 C15 X X X FW15 X X X TOTAL: 2 151 57 TOTAL: 0 146 64 SESSION 3 ID Avoids Escapes Failures ID Avoids Escapes Failures C1 8 7 0 FW1 0 5 10 C2 0 12 3 FW2 2 11 2 C3 0 14 1 FW3 0 15 0 C4 X X X FW4 0 14 1 C5 0 0 15 FW5 0 14 1 C6 1 14 0 FW6 0 15 0 C7 0 9 6 FW7 0 13 2 C8 0 6 9 FW8 0 13 2 C9 1 14 0 FW9 0 15 0 C10 0 7 8 FW10 0 14 1 C11 0 4 11 FW11 0 4 11 C12 1 14 15 FW12 0 5 10 C13 0 15 0 FW13 4 11 0 C14 0 6 9 FW14 0 15 0 C15 X X X FW15 X X X TOTAL: 11 122 77 TOTAL: 6 164 40 SESSION 4 ID Avoids Escapes Failures ID Avoids Escapes Failures C1 11 4 0 FW1 0 6 9 C2 0 12 3 FW2 3 12 0 C3 1 14 0 FW3 0 14 1 C4 X X X FW4 0 12 3 C5 0 0 15 FW5 0 14 1 C6 0 15 0 FW6 1 14 0 C7 0 7 8 FW7 2 13 0 C8 0 2 13 FW8 0 14 1 C9 1 14 0 FW9 2 13 0 C10 0 2 13 FW10 0 15 0 C11 0 3 12 FW11 0 2 13 C12 0 0 15 FW12 0 7 8 C13 1 13 1 FW13 3 12 0 C14 0 15 0 FW14 1 13 1 C15 X X X FW15 X X X TOTAL: 14 101 80 TOTAL: 12 161 37 SESSION 5 ID Avoids Escapes Failures ID Avoids Escapes Failures C1 11 4 0 FW1 0 2 13 C2 0 13 2 FW2 3 12 0 C3 4 11 0 FW3 1 14 0 C4 X X X FW4 0 13 2
59 C5 0 1 14 FW5 0 15 0 C6 1 14 0 FW6 1 14 0 C7 0 8 7 FW7 1 14 0 C8 0 2 13 FW8 0 1 5 0 C9 8 7 0 FW9 0 14 1 C10 0 7 8 FW10 0 15 0 C11 0 5 10 FW11 0 4 11 C12 0 0 15 FW12 0 3 12 C13 3 12 0 FW13 2 13 0 C14 0 15 0 FW14 1 13 1 C15 X X X FW15 X X X TOTAL: 27 99 69 TOTAL: 9 161 40 SESSION 6 ID Avoids Escapes Fa ilures ID Avoids Escapes Failures C1 11 4 0 FW1 0 2 13 C2 0 10 5 FW2 7 7 1 C3 4 11 0 FW3 1 13 1 C4 X X X FW4 0 15 0 C5 0 0 15 FW5 0 0 15 C6 3 12 0 FW6 8 5 2 C7 0 11 4 FW7 5 10 0 C8 0 2 13 FW8 0 12 3 C9 7 8 0 FW9 1 14 0 C10 12 3 0 FW10 0 15 0 C11 0 7 8 FW11 1 9 5 C12 0 0 15 FW12 0 12 3 C13 1 13 1 FW13 0 15 0 C14 1 14 0 FW14 2 12 1 C15 X X X FW15 X X X 39 95 61 TOTAL: 25 141 44 SESSION 7 ID Avoids Escapes Failures ID Avoids Escapes Failures C1 10 5 0 FW1 0 2 13 C2 0 15 1 FW2 5 10 0 C3 0 15 0 FW3 1 13 1 C4 X X X FW4 0 13 2 C5 1 6 8 FW5 0 0 15 C6 0 14 1 FW6 0 15 0 C7 0 12 3 FW7 0 12 3 C8 0 10 5 FW8 0 13 2 C9 0 15 0 FW9 1 14 0 C10 0 9 6 FW10 0 15 0 C11 0 5 10 FW11 0 10 5 C12 3 2 10 FW12 0 8 7 C13 0 13 2 FW13 0 15 0 C14 0 15 0 FW14 2 11 2 C15 X X X FW15 X X X 14 136 46 TOTAL: 9 151 50
60 SESSION 8 ID Avoids Escapes Failures ID Avoids Escapes Failures C1 9 6 0 FW1 0 5 10 C2 0 4 11 FW2 6 9 0 C3 0 15 0 FW3 0 1 5 0 C4 X X X FW4 0 13 2 C5 X 5 X FW5 0 2 13 C6 10 5 0 FW6 2 13 0 C7 0 2 13 FW7 2 8 5 C8 1 14 0 FW8 0 13 2 C9 0 8 7 FW9 3 12 0 C10 0 2 13 FW10 0 15 0 C11 0 6 9 FW11 0 11 4 C12 0 8 7 FW12 0 6 9 C13 1 0 14 FW13 1 14 0 C14 1 14 0 FW14 4 1 0 1 C15 X X X FW15 X X X 22 89 74 TOTAL: 18 146 46 SESSION 9 ID Avoids Escapes Failures ID Avoids Escapes Failures C1 9 6 0 FW1 0 5 10 C2 4 11 0 FW2 9 6 0 C3 1 14 0 FW3 2 13 0 C4 X X X FW4 0 11 4 C5 X X X FW5 0 0 15 C6 8 7 0 FW6 3 12 0 C7 0 2 13 FW7 2 8 5 C8 0 15 0 FW8 0 15 0 C9 0 11 4 FW9 0 15 0 C10 0 8 7 FW10 0 15 0 C11 2 11 2 FW11 0 4 11 C12 0 8 7 FW12 0 8 7 C13 2 13 0 FW13 1 14 0 C14 0 8 7 FW14 0 14 1 C15 X X X FW15 X X X 26 114 40 TOTAL: 17 140 53 SESSION 10 ID Avoids Escapes Failures ID Avoids Escapes Failures C1 12 3 0 FW1 0 3 12 C2 1 13 1 FW2 10 5 0 C3 4 11 0 FW3 0 14 1 C4 X X X FW4 0 8 7 C5 X X X FW5 0 0 15 C6 4 11 0 FW6 8 7 0 C7 0 13 2 FW7 2 7 6 C8 0 14 1 FW8 0 13 2 C9 0 2 13 FW9 2 13 0
61 C10 1 5 9 FW10 0 15 0 C11 0 9 6 FW11 0 5 10 C12 0 14 1 FW12 1 4 10 C13 1 14 0 FW13 1 14 0 C14 3 12 0 FW14 1 13 1 C15 X X X FW15 X X X TOTAL: 26 121 33 TOTAL: 25 121 64 SESSION 11 ID Avoids Escapes Failures ID Avoids Escapes Failures C1 15 0 0 FW1 0 8 7 C2 1 6 8 FW2 7 8 0 C3 2 12 1 FW3 2 13 0 C4 X X X FW4 0 13 2 C5 X X X FW5 0 0 15 C6 5 8 0 FW6 8 7 0 C7 0 12 3 FW7 3 4 8 C8 0 1 14 FW8 0 14 1 C9 0 5 10 FW9 0 15 0 C10 0 6 9 FW10 0 15 0 C11 1 11 3 FW11 0 4 11 C12 0 8 7 FW12 2 0 13 C13 4 8 3 FW13 0 15 0 C14 0 15 0 FW14 1 13 1 C15 X X X FW15 X X X TOTAL: 28 92 58 TOTAL: 23 129 58 SESSION 12 ID Avoids Escapes Failures ID Avoids Escapes Failures C1 13 1 1 FW1 0 7 8 C2 0 6 9 FW2 6 9 0 C3 1 13 0 FW3 2 13 0 C4 X X X FW4 0 13 2 C5 X X X FW5 0 0 15 C6 6 9 0 FW6 5 15 5 C7 0 15 0 FW7 2 2 11 C8 0 0 15 FW8 0 13 2 C9 0 3 12 FW9 3 12 0 C10 1 4 10 FW10 2 13 0 C11 0 9 6 FW11 0 8 7 C12 0 14 1 FW12 2 2 11 C13 1 10 4 FW13 2 13 0 C14 5 10 0 FW14 2 13 0 C15 X X X FW15 X X X TOTAL: 27 94 58 TOTAL: 26 133 51 SESSION 13 ID Avoids Escapes Failures ID Avoids Escapes Failures C1 9 6 0 FW1 0 9 6 C2 2 4 9 FW2 3 12 0
62 C3 0 8 7 FW3 4 11 0 C4 X X X FW4 0 13 2 C5 X X X FW5 0 0 15 C6 2 8 0 FW6 6 9 0 C7 1 13 1 FW7 6 6 3 C8 2 13 0 FW8 0 13 2 C9 0 7 8 FW9 1 14 0 C10 0 2 13 FW10 4 11 0 C11 0 10 5 FW11 0 3 12 C12 0 0 15 FW12 1 2 12 C13 2 11 2 FW13 4 11 0 C14 7 8 0 FW14 0 12 3 C15 X X X FW15 X X X TOTAL: 25 90 60 TOTAL: 29 126 55
63 References Anctil, M. (1989) Modulation of a rhythmic activity by serotonin via cyclic AMP in the coelenterate Renilla kllikeri. J. Comp. Physiol. B159; 4 91 500. Barker, E.L., Blakely, R.D., (1995). Norepinephrine and serotonin transporters: M olecular targets of antidepressant drugs. Psychopharmacology: The Fourth Generation of Progress Raven Press, New York, pp. 321 334. Barnes, N.M and Sharp, T. (1999 ). A review of central 5 HT receptors and their function. Neuropharmacology. 38:8; 1083 1152. Beyer C, Lin Q, Platt B, Malberg J, Hornby G, Sullivan K, Smith D, Lock T, Mitchell P, Hatzenbuhler T, Evrard A, Harrison L, Magolda R, Pangalos N, Schechter E, Rosenzweig Lipson S, Andree TH.(2009). Preclinical characterization of WAY 211612: a dual 5 HT uptake inhibitor and 5 HT (1A) receptor antagonist and potential novel antidepressant. British Journal of Pharmacology. 157(2):307 19. Boess F.G. and Martin I.L. (1994). Molecular Biology of 5 HT Receptors. Neuropharmacology 33; 275 317. Boutrel B, Monaca C, Hen R, Hamon M, and Adrien J. (2002). Involvement of 5 HT1A receptors in homeostatic and stress induced adaptive regulations of paradoxical sleep: s tudies in 5 HT1A knock out mice. Journal of Neuroscience 22; 4686 92. Buhot MC, Martin S, Segu L. (2000). Role of serotonin in memory impairment. Ann Med. 32(3); 210 21. Carlsson, A and Wong, D. (1997). A note on the discovery of selective serotonin reuptake inhibitors. Life Sciences 61(12):1203 Casto, E., Diaz, A, Rodriguez Gaztelumendi, Olmo, E., and Pazos, A. (2008). WAY100635 prevents the changes induced by fluoxetine upon the 5 HT 1A receptor functionality. Neuropharmacology. 55; 1391 1396. Chamberlain B., Ervin F.R., Pihl R.O., Young S.N. (1987). The effect of raising or lowering tryptophan levels on aggression in vervet monkeys Pharmacol Biochem Behav. 28(4); 503 10. Charney D. S. Woods S. W. Krystal J. H. & Heninger G. R. (1990) Serotonin function and human anxiety disorders. Annals of the New York Academy of Sciences 600 558 573.
64 Chemel, B.R., Roth, B.L., Armbruster, B., Watts, V.J. and Nichols, D.E. (2006). WAY 100635 is a potent dopamine D 4 receptor agonist. Psychopharm acology 188; 244 251. Journal of Neuroscience 6, pp 2427 2450. Chojnacka Wjcik E, Klodzinska A, Tatarczynska E. (2005). The anxiolytic like effect of 5 HT1B receptor ligands in rats: a possible mechanism of action. J Pharm Pharmacol. 57 (2); 253 7. Connections of the mesencephalic ventromedial tegmentum (VMT) in the cat. Modula tion of Sensorimotor Activity during Altera tions in Behavioral States New York: Alan R. Liss, pp. 39 73. Davidson C., Price G.W., Jones B.J., and Stamford J.A. (1997). (+) WAY 100135, a partial agonist, at native and recombinant 5 HT1B/1D receptors. Br J Pharmacol. 121 (4); 737 742. Erdtmann B D, Weber B, Schultze H J Vertebr Paleontol 20:394 399. Eriksson TM, Madjid N, Elvander Tottie E, Stiedl O, Svenningsson P, Ogren SO. (2008). Bloc kade of 5 HT 1B receptors facilitates contextual aversive learning in mice by disinhibition of cholinergic and glutamatergic neurotransmission. Neuropharmacology. 54(7); 1041 50. Erspamer, V and Asero, B (1953). Isolation of Enteramine From Extracts of P osterior Salivary Glands of Octopus Vulgaris and of Discoglossus Pictus Skin J. Biol. Chem ., Jan 1953; 200: 311 318 Feldman, J. M.; Lee, E. M. (1985). "Serotonin cont ent of foods: effect on urinary excretion of 5 hydroxyindoleacetic acid". Am. J. Clin Nutr 42 (4): 639 43. Fernandez Perez, Pache, and Sewell. (2005). Co administration of fluoxetine and WAY 100635 improves short term memory function. European Journal of Pharmacology 522; 78 83. Fernstrom, J.D and Wurtman, R. J. (1971). Brain serotoni n content: physiological dependence on plasma tryptophan levels. Science, 173; 149 152.
65 G. Kass Comparative Biochemistry and Physiology Part A: Molecular & I ntegrative Physiology Volume 146, Issue 1, pp. 9 25 G. Pinna, E. Costa and A. Guidotti. (2006). Fluoxetine and norfluoxetine stereospecifically and selectively increase brain neurosteroid content at doses inactive on 5 HT reuptake. Psychopharmacology 18 6; 362 372. Journal of Experimental Biology 182 265 270. He J, Hoffman SW, Stein DG. (2 004) Allopregnanolone, a progesterone metabolite, enhances behavioral recovery and decreases neuronal loss after traumatic brain injury. Restor Neurol Neurosci 22; 19 31. Heisler, L. K., Chu, H. M., Brennan, T. J., Danao, J. A., Bajwa, P., Parsons, L. H and Tecott, L. H. (1998). Elevated anxiety and antidepressant like responses in serotonin 5 HT1A receptor mutant mice. Proc Natl Acad Sci 95; 15049 15054. Hjorth S., Bengtsson H.J., Kullberg A., Carlzon D., Peilot H., Auerbach S.B. (2000). Serotonin autoreceptor function and antidepressant drug action. J.Psychopharmacol.,14 177 185. Hurley, IA; Mueller, RL; Dunn, KA; Schmidt, EJ; Friedman, M; Ho, RK; Prince, VE; scale for ray finned fish evolutio Proc Biol Sci .;274:489 498. IUPHAR DB: the IUPHAR database of G protein coupled receptors and ion channels. Nucl. Acids Res. 37 (Database issue): D680 D685 http://nar.oxfordjournals.org/cgi/content/full/37/suppl_1/D680",%20800,%20500 J. Puia, J.M. M ienville, K. Matsumoto, E. Costa and A. Guidotti. (2003). On the putative physiological role of allopregnanolone on GABA A receptor function, Neuropharmacology 44 ; 49 55. sy napses in the nerve net of Hydra J. Cell Biol. 51 (1971), pp. 318 323. Janvier, Philippe. 1997. Gnathostomata. Jawed Vertebrates. Version 01 January 1997 (under construction). http://tolweb.org/Gnathostomata/14843/1997.01.01 in The Tree of Life Web Pro ject, http://tolweb.org/ Johansson L, Sohn D, Thorberg SO, Jackson DM, Kelder D, Larsson LG, Rnyi L, Ross SB, Wallsten C, Eriksson H, Hu PS, Jerning E, Mohell N, Westlind Danielsson A. (1997). The pharmacological chara cterization of a novel selective 5
66 hydroxytryptamine1A receptor antagonist, NAD 299. J Pharmacol Exp Ther 283; 216 25. Kaufman, S. (1974). Properties of pterin dependent aromatic amino a cid hydroxylases. Ciba Foundation Symposium 22 Aromatic Amino Aci ds in the Brain pp 85 115 Kusserow, H., Davies, B., Hortnagl, H., Voigt, I., Stroh, T., Bert, B., Deng, D. R., Fink, H., Veh, R. W. and Theuring, F. (2004) Reduced anxiety related behaviour in transgenic mice overexpressing serotonin 1A receptors. Brain Res Mol Brain Res. 129; 104 116 Leathwood, P.D. 1987. Typtophan availability and serotonin synthesis. Proceedings of Nutrition Society 46; 143 156. Lenox, R and Frazer, H. (2002). Neuropsychopharmacology: The Fifth Generation of Progress American Co llege of Neuropsychopharmacology. Ch: 79:1149 1163. Long A.B., Kuhn C.M. and Platt M.L. (2009 ). Serotonin shapes risky decision making in monkeys. Social Cognitive and Affective Neuroscience. E publication ahead of print. John Hopkins University Press Baltimore and London. Martel JC, Leduc N, Ormire AM, Faucillon V, Danty N, Culie C, Cussac D, and Newman Tancredi A. WAY 100635 has high selectivity for serotonin 5 HT(1A) versu s dopamine D(4) receptors. Eur J Pharmacol 574; 15 9. Meltzer, H.Y (1990). Role of Serotonin in Depression. Annals of the New York Academy of Sciences. 600; 486 499. Mendelsohn D, Riedel WJ, Sambeth A. (2009) Effects of acute tryptophan depletion on memory, attention and executive functions: a systematic review. Neurosci Biobehav Rev. 33(6); 926 52. Monti, J. M. and Jantos, H. (2004) Effects of the 5 HT1A receptor ligands flesinoxan and WAY 100635 given systemically or microinjected into the latero dorsal tegmental nucleus on REM sleep in the rat. Behav Brain Res 151 ; 159 166. Nelson C. J. Jordan W. P. & Bohan R. T. (1997). Daily fluoxetine administration impairs avoidance learning in the rat without altering sensory thresholds. Progress in Neuropsychopharmacology and Biological Psychology 21; 1043 1057. Nichols, D. (2004). Hallucinogens. Pharmacology & Therapeutics 101 (2): 131 81
67 Ogren SO, Eriksson TM, Elvander Tottie E, D'Addario C, Ekstrm JC, Svenningsson P, Meister B, Kehr J, St iedl O. (2008). The role of 5 HT(1A) receptors in learning and memory. Behav Brain Res. 195(1); 54 77. Page, I.H. (1968). Serotonin. Chicago, Yearbook Medical Publishers. Parks, C. L., Robinson, P. S., Sibille, E., Shenk, T. and Toth, M. (1998). Increase d anxiety of mice lacking the serotonin1A receptor. Proc Natl Acad. 95; 10734 10739. Pattij T, Broersen LM, van der Linde J, Groenink L, van der Gugten J, Maes RA, Olivier (2004). Operant learning and differential reinforcement of low rate 36 s respondin g in 5 HT1A and 5 HT1B receptor knockout mice. Behav Brain Res. 141(2); 137 45. Pinna G, Agis Balboa RC, Pibiri F, Nelson M, Guidotti A, Costa E. (2008). Neurosteroid biosynthesis regulates sexually dimorphic fear and aggressive behavior in mice. Neuroc hem Res. 33(10); 1990 2007. Pinna, G., Costa, E., and Guidotti, A., (2009). SSRIs act as selective brain seroidogenic stimulants (SBSSs) at low doses that are inactive on 5 HT reuptake. Current Opinion in Pharmacology 9; 24 30. Ramboz, S., Oosting, R. Amara, D. A., Kung, H. F., Blier, P., Mendelsohn, M., Mann, J. J., Brunner, D. and Hen, R .(1998) Serotonin receptor 1A knockout: an animal model of anxiety related disorder. Proc Natl Acad Sci 95; 14476 14481. Rapport MM, Green AA, Page IH (1948a): P artial purification of the vasoconstrictor in beef serum. J Biol Chem; 174:735 738 Rapport MM, Green AA, Page IH (1948c): Crystalline Serotonin. Science 108:329 330 Rapport MM, Green AA, Page IH. (1948b) Serum Vasoconstrictor (Serotonin). III. Chemical Inactivation J. Biol. Chem ; 176: 1237 1241 Romeo, Cheny, Zivkovic, Costa, and Guidotti. (1994). Mitochondrial diazepam binding inhibitor receptor complex agonists antagonize dizocilpine amnesia: putative role for allopregnanolone. American Society for Pharmacology and Experimental Theraputics 270; 89 96. Sarnyai, Z., Sibille, E. L., Pavlides, C., Fenster, R. J., McEwen, B. S. and Toth, M. (2000). Impaired hippocampal dependent learning and functional abnormalities in the hippocampus in mice lacking serotonin(1A) receptors. Proc Natl Acad Sci 97 ; 14731 14736.
68 Schechter LE, Smith DL, Rosenzweig Lipson S, Sukoff SJ, Dawson LA, Marquis K, Jones D, Piesla M, Andree T, Nawoschik S, Harder JA, Womack MD, Buccafusco J, Terry AV, Hoebel B, Rada P, Kelly M Abou Gharbia M, Barrett JE, and Childers W. (2005). Lecozotan (SRA 333): a selective serotonin 1A receptor antagonist that enhances the stimulated release of glutamate and acetylcholine in the hippocampus and possesses cognitive enhancing properties J Pharmacol Exp Ther 314(3); 1274 89. Simonenkov A.P. and Fedorov VD. (2002). Serotonin and serotonin receptors in the genesis of stress and adaptation Vestn Ross Akad Med Nauk 8; 9 13. Sitaraman, Zars, LaFerriere, Chen, Sable Smith, Kitamoto, Rottingh aus, and Zars, Drosophila Sci 105(14) pp. 5579 5584 Soares, J.C, and Gershon, S. (2000). Bipolar Disorders: Basic Mechanisms and Therapeutic Implications. Informa Health Care ISBN 082 470360X. Ch: 7:121 125. Sprouse J, Reynolds L, Li X, Braselton J, Schmidt A (January 2004). 8 OH DPAT as a 5 HT7 agonist: phase shifts of the circadian biological clock through increases in cAMP production. Neuropharmacology 46; 52 62. Stahl, S.M. (1999 ). Mechanism of action of serotonin selective reuptake inhibitors: Serotonin receptors and pathways mediate therapeutic effects and side effects. Journal of Affective Disorders 53(3); 215 235. Steiger, H. (2004). Eating disorders and the serotonin conn ection: state, trait and developmental effects. J Psychiatry Neurosci. 29(1); 20 9. Stensrud T, Ingjer F, Holm H, Strmme S.B. (1992). L tryptophan supplementation does not improve running performance. Int J Sports Med. 13(6); 481 5. Stone J., Dreher edit ion Sydney: Maitland Publications Tatarczynska E, Klodzinska A, Stachowicz K, Chojnacka Wjcik E. (2004). Effects of a selective 5 HT1B receptor agonist and antagonists in animal models of anxiety and depression. Behav Pharmacol. 15 (8); 523 34. Thompson, C. (2002). Onset of action of antidepressants: results of different analyses. Human Psychopharmacology: Clinical and Experimental. 17:S1, S27 S32.
69 Trk, I. (2006). Anatomy of the Serotonergic System. The Neuropharmacology of Serotonin. 600:9 34. Twarog, B and Page, IH. (1953) Serotonin Content of Some Mammalian Tissues and Urine and a Method for Its Determination. Am J Physiol 175: 157 161, 1953 V. Uzunova, Y. Sheline, J.M. Davis, A. Rasmusson, D.P. Uzunov, E. Costa and A. Guidotti. (1998). Increase in the cerebrospinal fluid content of neurosteroids in patients with unipolar major depression who are receiving fluoxetine or fluvoxamine, Proc Natl Acad Sci. 95 ; 3239 3 244. Hydrobiologia 530/531 pp. 135 144. Valluzi, J.A and Chan, K. (2007). Effects of fluoxetine on hippocampal dependent and hippocampal independent learning tasks. Behavioral Pharmacology. 18; 507 13. Weiger, WA. (1997). Serotonergic Modulation of Behavior: A Phylogentic Overview. Biol. Rev. 72 pp. 61 95. Whitaker Azmitia, J.M (1999). The Discovery of Serotonin and its Role in Neuroscience. Neuropsychopharmacology Vol. 21 No. 2S Winde r VL, Sapozhnikova Y, Pennington PL, Wirth EF. (2008). Effects of fluoxetine exposure on serotonin related activity in the sheepshead minnow (Cyprinodon variegatus) using LC/MS/MS detection and quantitation. Comp Biochem Physiol C Toxicol Pharmacol. 149(4 ); 559 65. Wong D, Bymaster F, Engleman E. (1995). Prozac (fluoxetine, Lilly 110140), the first selective serotonin uptake inhibitor and an antidepressant drug: twenty years since its first publication. Life Sciences. 57(5):411 41 Woolley and Shaw, (1954). A Biochemical and Pharmacological Suggestion about Certain Mental Disorders. Pro c Natl Acad Sci U S A. 40 (4) : 228 231. Yasuno F, Suhara T, Nakayama T, Ichimiya T, Okubo Y, Takano A, Ando T, Inoue M, Maeda J, Suzuki K. (2003) Inhibitory effect of hippocampal 5 HT1A receptors on human explicit memory. Am J Psychiatry. 160(2); 334 40. Young S.N and Leyton M. (2002). The role of serotonin in human mood and social interaction: Insight from altered tryptophan levels. Pharmacolog y, Biochemistry and Behavior. 71; 857 865 Young S.N., Pihl R.O., and Ervin FR. (1988). The effect of altered tryptophan levels on
70 mood and behavior in normal human males. Clinical Neuropharmacology. 1; 207 15.