\documentclass[12pt, letterpaper, twocolumn]{report}
\usepackage{fullpage}
\usepackage[pdftex,hypertexnames=false,linktocpage=true]{hyperref}
\hypersetup{colorlinks=true,linkcolor=blue}
\usepackage{etoolbox}
\begin{document}
\title{Effects of Anxiolytic and Anxiogenic Drugs on Zebrafish}
\author{RPBruiser}
\date{April 13, 2015}
\maketitle
\begin{abstract}
The zebrafish (\textit{Danio rerio}) have become a prominent organism in studies that model anxiety and stress. Here I further investigate and zebrafish models of anxiety and stress by analyzing how anxiolytic and anxiogenic drugs affect the behaviors of the zebrafish. Experimental manipulation includes acute exposure to  Librium (Chlordiazepoxide), Ativan (Lorazepam), Caffeine, and Nicotine. Using Fiji, an ImageJ extension, the movements of the zebrafish were tracked while introduced to the novel tank paradigm, as well as to a variety of anxiolytic and anxiogenic drugs.
\end{abstract}
\renewcommand{\chaptername}{}
\chapter{Introduction}
\indent A central question surrounding pharmacuticals, and drugs in general, today is how they effect mood and behavior. Through mutliple behavior paradigms and nueroimaging, animals have been proven to be a useful model for studying anxiety and its behavioral effects\cite{label4}. Zebrafish( \textit{Danio rerio}) have been used for modeling behavior in biomedical research. Zebrafish have all of the classic neurotransmitters found in vertebrates, andt it has been proven that their neuroendocrine system provides a stron physiological response to stress. More importantly, zebrafish have been shown to be physiologically homologous to huumans.\cite{label1}\cite{label2}\cite{label4}\\
\indent As seen in other species, being introduction to novelty causes an anxiety response in zebrafish. Zebrafish are introduced to novelty through what is called a novel tank test, which is very similar to the open field test used on rodents\cite{label1}. Using this model, researchers are able to analyze different behavioral parameters, including latency, transitions into higher regions of the tank, erratic movements, and freezing spells, and asses the anxiety. Usualy the less time spent in higher levels of the tank, the longer latency to enter the higher levels, increased erraticity of movement and freezing speels are attributed to increased anxiety \cite{label1}\cite{label2}\cite{label3}.\\
\indent Besides novelty, another way to invoke anxiety-like response is to use various anxiolytic and anxiogenic drugs. Studies have already examined axiety in zebrafish using a variety of anxiolytic drugs such as nicotine, $\alpha$-fluromethylhistidine, ethanol, and diazepm \cite{label1}\cite{label2}\cite{label4}.\\
\indent However, novelty is not the only way to induce anxiety-like behaviors. Here I asses the effects of anxiolytic and anxiogenic on zebrafish by comparing how the behavior of zebrafish is effected when introduced to certain drugs. Specifically I aim to test the behavioral responses to two anxiolytic and two anxiogenic drugs, and to obtain and analyze the responses to said drugs and compare these responses to the known responses associated with anxiety-like behavior \cite{label1}\cite{label2}\cite{label4}. The behaviors that will be monitored throughout this expirement include: latency  latency between regions, time spent in the upper regions of the tank, number of transitions to the upper regions, number of erratic movements, and number of freezing spells. Experimental manipulation include acute exposure to librium (chlordiazepoxide), ativan (lorazepam), caffeine, and nicotine.\\
\renewcommand{\chaptername}{}
\chapter{Methods}
\section{Animals and Housing}
Total of 4 adult (age) male and female zebrafish were taken from the Biology Department. This study used a single type (wild-type) zebrafish strain (n = 5). The fish being used were already acclimated to the labratory settings and housed in a group of 14 fish in a 11-G tank. Illumination was provided by 36 ceiling-mounted fluorescent ligh tubes, as well as ambient light from sun coming through the windows. The fluorescent light tubes were on an alternating schedule of roughly 50 minutes. This 50 minute cycle was irregular during the week, but was consistent on a week to week basis. All the fish used had never been used in expirements before.
\section{Novel Tank Test}
After treatment, the zebrafish were placed indivdually into a 1.5-L tank \{15.2 height x 27.9 top x 22.5 bottom x 7.1 width cm\} obtained from Aquatic Habitats\textsuperscript{\textregistered} filled with tank treated water. The novel tank was set on a flat, level, and stable surface. It was divided into three equal horizontal sections, marked by a dividing line drawn on the outside of the walls. Once the zebrafish were transfered into the novel tank, the were given an acclimation period of 2 minutes. There behavior and motion was observed and tracked using (name of tracking software) over an 8 minute period. The following behaviours were recorded: latency between regions, time spent in the upper regions of the tank, number of transitions to the upper regions, number of erratic movements, and number of freezing spells. Erratic movement was defined as any sudden change in velocity or direction, or rapid darting behavior. Freezing spells were defined as the lack of movement in zebrafish, excluding the gills and eyes, that lasted longer than 1 second. Longer latency and freezing spells, fewer transitions to upper regions, and increased erraticity in movements are behaviors that are indicative of high stress and anxiety in zebrafish. These behavioral aspects were chosen as they have been proven in previous studies \cite{label2}.
\section{Drug Induced Anxiety}
For drug exposure expirements, zebrafish were extracted from there home tank and placed in a seperate holding tank. They were acutely dose by being placed in AMOUNT Librium (PERCENTAGE),  AMOUNT Ativan (PERCENTAGE), AMOUNT Caffeine(PERCENTAGE), and AMOUNT Nicotine(PERCENTAGE), and were left for 5 minutes. The acutely exposed fish were imidiately tested in the novel tank test. There were four fish (n = 4) in this study. The control fish (n = 1) was also placed in a seperate holding tank of fish-grade water for five minutes, and imidiately tested in the novel tank test. In all expirements, the novel tank test was performed in a nove tank containing fish-grade water.
\chapter{Results}
\makeatletter
\patchcmd{\chapter}{\if@openright\cleardoublepage\else\clearpage\fi}{}{}{}
\makeatother
\chapter{Discussion}
\onecolumn
\begin{thebibliography}{10}
\addtolength{\leftmargin}{0.2in}
\setlength{\itemindent}{-0.2in}
\bibitem{label1} Egan, R.J., Bergner, C.L., Hart, P.C., Cachat, J.M., Canavello, P.R., Elegante, M.F., and Kalueff, A.V., ``Understanding behavioral and physiological phenotypes of stress and anxiety in zebrafish," \emph{Behavioural Brain Research}, pp. 38-44, June 2009.
\bibitem{label2} Haymore, W., ``Zebrafish Behavioural Response to Chronic Drug Exposure," \emph{Tulane University Medical School: Department of Pharmocology}, June 2009.
\bibitem{label3} Rosemburg, D.B., Mussulini, B.H.M., Piato, \^A.L., Calcagnotto, M.E., Bonan, C.D., and de Oliveria, D.L., ``Differences in spatio-temporal behavior of zebrafish in the open tank paradigm after a short-period confinement into dark and bright environments," \emph{PLoS ONE}, October 2011.
\bibitem{label4} Stewart, A., Gaikwad, S., Kyzar, E., Green, J., Roth, A., and Kalueff, A.V., ``Modeling anxiety using adult zebrafish: A conceptual review," \emph{Neuropharmacology}, pp. 135-143, July 2011.
\end{thebibliography}
\end{document}