Thesis Proposal Draft - Gemstone Program

Running Head: VALIDATING AN ANIMAL MODEL OF ADHD: MEASURING

IMPULSIVITY AS CORRELATED WITH dPL ACTIVITY AND MEDIATED BY

ADDERALL

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ADMINISTRATION IN FETAL NICOTINE RATS

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APA 6th

Validating an Animal Model of ADHD: Measuring Impulsivity as Correlated with dPL Activity and Mediated by Adderall

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Administration in Fetal Nicotine Rats

Team Research in Testing ADHD’s Link in Neuroscience (RITALIN)

Team Research Proposal

Brian Barnett, Valerie Cohen, Taylor Hearn, Emily Jones, Reshma Karilyl, Alice Kunin, Sen

Kwak, Jessica Lee, Brooke Lubinski, Gautam Rao, Ashley Zhan

The University of Maryland, Gemstone Program

VALIDATING AN ANIMAL MODEL OF ADHD: MEASURING IMPULSIVITY AS

CORRELATED WITH dPL ACTIVITY AND MEDIATED BY ADDERALL

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Table of Contents

Abstract ...........................................................................................................................................

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Introduction ....................................................................................................................................

4

Research Questions & Hypotheses ..............................................................................................6

Implications ..................................................................................................................................6

Literature Review ..........................................................................................................................

7

Clinical Components ....................................................................................................................7

Fetal Nicotine Rat Model .............................................................................................................9

Stop-Signal Task ........................................................................................................................11

Neurophysiology ........................................................................................................................12

Pharmacology .............................................................................................................................13

Methodology .................................................................................................................................

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Research Design .........................................................................................................................15

Pilot Study ..................................................................................................................................16

Adderall

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Study.........................................................................................................................17

Neural Recordings ......................................................................................................................19

Fetal Nicotine Study ...................................................................................................................19

Data Analysis .............................................................................................................................19

Histology ....................................................................................................................................20

Animal Care ...............................................................................................................................20

Study Limitations .......................................................................................................................21

Conclusion ....................................................................................................................................

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Appendix A: Anticipated Budget ...............................................................................................

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Appendix B: Timeline for Team Success ...................................................................................

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Appendix C: Schoenbaum Electrode Construction ..................................................................

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Appendix D: Histology with Nissl Staining ...............................................................................

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Appendix E: Glossary of Terms .................................................................................................

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References .....................................................................................................................................

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Abstract

Currently, the neurological basis of attention deficit hyperactivity disorder (ADHD) is not well

3 established; this disorder is diagnosed behaviorally rather than quantitatively. We seek to measure neural firing in the dorsal prelimbic cortex (dPL) to determine whether it is associated with an increase in impulsivity observed in the fetal nicotine rat model during the stop-signal task. We will also establish whether Adderall

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administration alters dPL neural firing and improves stop-signal task performance of this animal model. From these findings, we will determine the validity of fetal nicotine rats as models of ADHD. These results will allow the fetal nicotine rat model to be used to further study the neurological basis of ADHD.



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Validating a Model of ADHD: Measuring Impulsivity as Correlated with dPL Activity and

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Mediated by Adderall Administration in Fetal Nicotine Rats

ADHD is characterized by impulsivity and hyperactivity that influences one’s ability to concentrate and regulate behavior (National Institute of Mental Health, 2007). These symptoms usually appear in early stages of life, and in many cases, they last through adulthood. Children with ADHD are more likely to encounter academic difficulties, including but not limited to scoring poorly on exams and withdrawing prematurely from school. Recent research predicts that ADHD affects approximately five to ten percent of school aged children. (Evans, Morrill, &

Parente, 2011).

This disorder has been deemed highly controversial due to the disagreements over its diagnostic criteria, its frequency of diagnosis, and its method of treatment. Currently there is no well-established and experimentally verified neurological basis for ADHD, and thus the disorder has been diagnosed based on behavioral observations instead of quantitative measures. This insufficient method of diagnosing ADHD has led to numerous misdiagnoses, rising medical costs, and incorrect medication prescriptions, which can be detrimental to the health of patients because of possible harmful side effects. Focused research in the neural basis of ADHD will help create a concrete method for diagnosing this disorder and how to treat it. Once a neural basis is established, doctors could use functional magnetic resonance imaging (fMRI) brain scans to determine if the brain is malfunctioning and if the child has a medical disorder. This would be more accurate than diagnosing the disorder based on behaviors and qualitative observations, benefiting health professionals, patients, and their families (Zwi, Ramchandani, & Joughin,

2000).



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Research demonstrates that ADHD is linked to failure of the brain to control or inhibit

5 behavior. The stop-signal task, a popular method used in neuroscience and psychology to measure impulsivity, has shown that those with ADHD tend to have slower inhibition response times (Eagle & Baunez, 2010). These tasks were correlated with activity in the prefrontal cortex, notably the dPL, suggesting an association of the prefrontal cortex with response inhibition and modulation systems (Aron & Poldrack, 2004; Bari et al., 2011). Experimentally, the stop-signal task is used to observe behavior and determine neural activity related to impulsive action.

Numerous animal models have been established for ADHD; however, the validity of these models remains debatable. Currently, one possible model for ADHD is the fetal nicotine rat. These rats model the relationship noted between pregnant mothers who smoke and the increase in children exhibiting ADHD behaviors (Wasserman, Liu, Pine, & Graziano, 2001).

Research has demonstrated that fetal exposure to nicotine leads to a dysfunction in the development of dopaminergic and noradrenergic pathways in the brain; this dysfunction has been attributed to notable decreases in attention span and increases in impulsivity (Muneoka et al.,

1997). Because the complete validity of the fetal nicotine rat model has not been proven yet, our study would serve to confirm or disprove the fetal nicotine rat as a plausible model of ADHD.

In order to account for these neurological differences, various pharmacological treatments in addition to behavioral therapies are given to patients to cope with their symptoms.

One notable treatment is Adderall

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, an amphetamine that has been shown to reduce ADHD symptoms in patients (Weisler, 2005). The relationship between Adderall

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and its impact on neurotransmitters is known; however, its impact on neural firing in relation to impulsivity is yet unexamined. Neural firing in a specific brain region during a task demonstrates that the region is involved in regulating the response. Validating the fetal nicotine rat model could be



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ADMINISTRATION IN FETAL NICOTINE RATS accomplished by linking Adderall

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administration with improved performance on the stop-signal task and altered neural firing in the dPL in the model.

In this study, we will examine this relationship in order to better validate the fetal nicotine rat model. Is there a correlation between patterns of neural firing in the dPL and variations in impulsivity in control rats? Is this firing disrupted and impulsivity increased in fetal nicotine rats? Will administration of Adderall

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alter dPL firing and improve stop-signal task performance in fetal nicotine rats? We hypothesize that patterns of neural firing in the dPL in control rats will reflect the role of the dPL as an impulsivity control area. In addition, we hypothesize the fetal nicotine rats will show reduced neural firing in the dPL in response to the stop-signal and increased impulsivity. Finally, we hypothesize that Adderall

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administration will increase dPL neural firing and decrease stop-signal reaction time (SSRT) in rats that exhibit increased impulsivity.

In recent years, the dramatic increase in the number of ADHD diagnoses is attributed to general qualitative observations of an individual’s behavior; the lack of a clinically significant and verified neurological basis has resulted in significant incidences of misdiagnoses (Kim &

Miklowitz, 2002). Understanding the neurological regions associated with the pathology of

ADHD would be instrumental in diagnosing patients in a consistent and empirical manner.

Research into the role of the dPL in ADHD could formulate a concrete diagnosis of the disorder, which may reduce incidences of misdiagnosis.

If we find that abnormal neural firing in the dPL is correlated with impulsivity in fetal nicotine rats, this will further validate the fetal nicotine rat as an acceptable animal model of

ADHD, as the dPL is a homologous brain area, or one shared by humans and rats. After analyzing the association of the dPL and impulsivity, we will determine whether Adderall

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ADMINISTRATION IN FETAL NICOTINE RATS administration increases neural firing in the dPL, which is associated with improved task performance, in response to the stop-signal, which will elucidate the causal relationship between

7 alterations in neural firing in the dPL and increased impulsivity in individuals with ADHD.

Proving this relationship would be fundamental for health professionals and pharmaceutical companies because they would have an empirical basis of diagnosis and could develop treatments to target these brain areas.

Literature Review

In order to find a neural basis of ADHD, we must choose a valid rat model of the disorder and integrate it with Adderall

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administration in addition to neural recording of the dPL. We begin with an overview of the clinical components of ADHD to assess the deficiencies in the current system of diagnosis. We also analyze the fetal nicotine rat model, which has been shown to represent ADHD symptoms but requires further study to validate it as a model. We will then examine relevant research on neurophysiology of ADHD, specifically that which concerns. The dopamine and noradrenaline pathways and the dPL are the focus of our neurological research. In addition to a physiological basis, we will also examine the multiple behavioral factors of ADHD, one of which is response inhibition. Inhibition of an already-initiated response is repressed in

ADHD patients and causes impulsive behavior. The stop-signal task is a strong measure of both inhibition and impulsivity. Finally, we examine Adderall

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, a proven treatment for ADHD inhibits dopamine and noradrenaline reuptake, which then reduces impulsivity.

Clinical Components

The Diagnostic and Statistical Manual-IV (DSM-IV) is the American Psychiatric

Association’s most recently produced guide for the clinical diagnosis of ADHD. According to



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ADMINISTRATION IN FETAL NICOTINE RATS the manual, the most common symptoms associated with the disorder are impulsivity, inattention, and hyperactivity (American Psychiatric Association, 1994).

Past studies suggest that the development of ADHD can be attributed to both genetic and environmental factors (Slaats-Willemse, Swaab-Barneveld, de Sonneville, van der Meulen, &

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Buitelaar, 2003). Prenatal exposure to teratogens, factors which affect fetal development, such as alcohol and drugs often increases the chance of developing ADHD and often exacerbates

ADHD-like symptoms in children. Children who have been prenatally exposed to alcohol, for instance, have been shown to display symptoms associated with ADHD such as hyperactivity, impulsivity, and disruptive behavior. Similarly, the children of mothers who smoked tobacco during pregnancy had 2.7-fold increase in risk for developing ADHD (Curatolo, D’Agati, &

Moavero, 2010).

Recently, there has been a significant increase in the number of diagnoses of ADHD.

From 2003 to 2007, there was a 21.8% increase in the reported incidence of ADHD among children/adolescents between the ages of four and seventeen (Center for Disease Control and

Prevention). Although some of these diagnoses are accurate, many are believed to be incorrect.

This is due to the fact that current methods of diagnosing ADHD as outlined by the DSM-IV rely solely on behavioral observations. Furthermore, these guidelines do not consider that individuals within a specific subtype can have symptoms, which vary in severity. Certain factors, such as gender, age, and cultural background must also be taken into account when making the diagnosis

(Frick & Nigg, 2011). The establishment of a neurological basis of ADHD would greatly enhance the accuracy of diagnosing the disorder. Furthermore, treatment administered to patients would be more effective in controlling ADHD if it is able to target the specific region of the brain monitoring the exhibition of ADHD symptoms.



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Fetal Nicotine Rat Model

The fetal nicotine rat model serves to represent various ADHD symptoms and brain

9 deficiencies similar to those found in humans. In our particular study, the fetal nicotine rat model involves rats whose mothers were administered nicotine while pregnant. This section of the literature review explores different rat models that have been used in previous studies and further attempts to show the validity of the fetal nicotine rat model.

Sontag, Tucha, Walitza, and Lange found that the best animal model should combine face validity, construct validity, and predictive validity (2010). Face validity is based primarily on similarities in symptoms; therefore, a good animal model should demonstrate three core symptoms of ADHD to be present: attention deficit, hyperactivity, and impulsivity. Most models are based on similarities in symptoms alone. However, this type of validity could be misleading because not every hyperactive rat may have ADHD. Sontag et al. also found that construct validity shows that the model corresponds to an established pathophysiological basis of the disorder. This type of validity is more highly emphasized than face validity because it connects the behavioral symptoms with the modeled disease. In addition, predictive validity is the ability to predict unknown characteristics of the neurobiology and pathophysiology of a disorder to provide potential new treatments. In order to analyze this type of validity, drugs with similar effects in humans and the animal model are used to validate the model. Sontag et al. analyzed the spontaneously hypertensive rat (SHR), one example of a valid rat model that shows several aspects of face, construct, and predictive validity; however, because these rats are bred for hypertension, this symptom serves as a confounding variable because it is not present in ADHD, which limits the construct validity of this model. According to Sontag et al., even though there



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10 are many different animal models that have been used to study ADHD, no model has shown all three types of validity that are not limited by potential confounding variables.

Although a thoroughly validated animal model of ADHD has not yet been established, several studies have shown that damage to areas of the brain involved in behavioral inhibition, such as the prefrontal cortex, during fetal development may be linked to behavioral problems.

These behavioral problems are prevalent in the children of mothers who smoke tobacco during pregnancy and in controlled fetal nicotine trials, which suggests a causal link between developmental nicotine exposure and impulsivity (Wasserman et al., 2001). This provides evidence that the fetal nicotine rat model could be a valid animal model of ADHD. Nicotine targets certain neurotransmitter receptors in the fetal brain, which leads to deficits in the number of brain cells, eventually creating changes in synaptic activity (Slotkin, 1998). Specifically, prenatal nicotine exposure has been shown to lead to dysfunctions in the development of dopaminergic and noradrenergic systems in the rat brain (Muneoka et al., 1997). Dysfunctions in the noradrenergic system in the brain can severely decrease attention and focus, which are key components of ADHD and measures of construct validity. This rat model also is relevant to humans, especially mothers who smoke during pregnancy. Children between 12-17 years old whose mothers reported smoking more than half a pack of cigarettes daily during pregnancy were found to be significantly more likely to be diagnosed with conduct disorder, which is a broad disorder that is defined as having defiant or impulsive behavior, a key symptom of ADHD

(Thapar et al., 2003); these behavioral symptoms are also present in the fetal nicotine model, demonstrated face validity. Because the fetal nicotine rat model is not heavily used in research that examines neural and behavioral correlates of abnormal impulsivity, its predictive validity as a model of ADHD has not been established. Our study aims to validate or invalidate the fetal



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11 nicotine rat model based on its predictive validity. In addition, we will further establish construct validity, as the neurophysiology of changes due to maternal smoking has not been established.

Importantly, even if this model does not fully conform to a model ADHD, this research will provide important information related to deficits observed in children prenatally exposed to nicotine.

Stop-Signal Task

In clinical studies, the stop-signal task is a procedure that is generally used to measure impulsivity. The task gauges how quickly an initiated response is inhibited (Eagle & Baunez,

2010). We will use the stop-signal task because it provides a quantitative measure of motor inhibition by examining the SSRT and the stop accuracy. The SSRT is the time needed by the rat to inhibit an initiated response, and the stop accuracy is the percent of stop trials during which the subject correctly inhibits a response (Bari et al., 2011). By obtaining and analyzing a subject’s SSRT values upon completion of the stop-signal task, it is possible to use these values as a basis for measuring inhibitory control and impulsivity, as a longer SSRT and lower stop accuracy would indicate greater impulsivity and lower inhibitory control.

Experts in the fields of clinical psychology and psychopathology have made extensive use of SSRTs to study response inhibition in persons deemed to be generally impulsive, such as those with ADHD. Elevated SSRTs, which suggest that a greater amount of time is needed to inhibit an initiated response, have been associated with children and adult ADHD patients. This relationship has also been demonstrated in animal models of ADHD. A high SSRT value, therefore, is correlated to a lower level of inhibitory control and higher level of impulsivity

(Verbruggen & Logan, 2009).

Neurophysiology



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The prelimbic cortex is a subsection of the prefrontal cortex in both human and rat brains that functions as an executive control center. Projection sites, which are destinations for neural activity, of the prelimbic cortex, include the insular cortex, claustrum, nucleus accumbens, thalamus, amygdala, and brainstem. The prelimbic cortex is highly correlated with cognitive processing; subsequently, its lesioning has significant influence on motor and behavioral tasks

(del Campo, Chamberlain, Sahakian, & Robbins, 2011).

Neurotransmitters play key roles in information processing within brain structures such as the prelimbic cortex. The dopaminergic and noradrenergic neurotransmitter pathways are both integral to the control of prefrontal-dependent cognitive processes such as behavioral inhibition and impulsivity. For example, ADHD patients have decreased dopamine and noradrenaline activity in fronto-striatal circuits (del Campo et al., 2011). This decrease results from the potential combination of imbalances in neurotransmitter synthesis, release, receptor activation, and neuronal responsiveness . Research shows that ADHD patients with difficulty controlling their inhibitions have malfunctioning noradrenergic receptors while those with increased impulsive behavior have malfunctioning dopamine receptors (van Gaalen, van Koten,

Schoffelmeer, & Vanderschuren, 2006).

The dPL is a region in the prelimbic cortex and is the focus of our research (see Figure

1). Inactivation of the dPL, such as by lesioning the region, is correlated with slower SSRTs and mediates the effects of atomoxetine on improving SSRTs. When atomoxetine is used to locally block the noradrenaline reuptake in the dPL, stop-signal task performance improves. While dPL inactivation reduces the stop-signal reaction time of rats and their stop accuracy, it does not have control over the go reaction time and go accuracy. The prelimbic cortex affects performance on behavioral tasks, and lesioning prelimbic cortex impairs rats from making decisions associated



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ADMINISTRATION IN FETAL NICOTINE RATS with these tasks. Therefore, this region likely controls action selection, planning, and selective

13 attention. Deficiency in the evaluation of the task components results in an inability to choose between two actions. This inhibition is a phenomenon that dPL-lesioned rats have demonstrated in previous research (Bari et al., 2011). The dPL is a strong choice for study because it has been implicated in impulsivity and ADHD and is present in both rats and humans, yet we do not know how neural firing in the dPL might change in rats exposed to prenatal nicotine or Adderall

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Figure 1. Rat brain coronal cross section with the dPL circled (Eagle & Baunez, 2010). The areas of the brain shown (from left to right): pre-genual cingulated cortex (CG), prelimbic cortex

(PL), infralimbic cortex (IL), orbitofrontal cortex (OF), dorsomedial striatum (DMStr), dorsolateral striatum (DLStr), nucleus accumbens core (NAcbC), nucleus accumbens shell

(NAcbS), and subthalamic nucleus (STN).

Pharmacology

Various pharmacological treatments in addition to behavioral therapies are administered to patients to manage ADHD symptoms. The three main chemical components of ADHD pharmacological treatments are atomoxetine, methylphenidate, and amphetamine (Bari et al.,

2011). The most common treatment for ADHD in patients is amphetamine stimulant



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ADMINISTRATION IN FETAL NICOTINE RATS administration, with mixed amphetamine salts being the most effective. Amphetamines have

14 been shown to effectively reduce hyperactivity, impulsivity, and inattention in patients with

ADHD (Weisler, 2005).

Adderall

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is composed of two different enantiomers of amphetamine: D-amphetamine and L-amphetamine. It consists of a combination of four amphetamine salts: D-amphetamine saccharate, D,L-amphetamine aspartate, D-amphetamine sulfate, and D,L-amphetamine sulfate at equal weights. This mixture gives an approximate ratio of 75-80% D-amphetamine and 20-

25% L-amphetamine in a 10mg tablet. It is hypothesized that this salt combination contributes to the extended release of the drug due to differential absorption by the body of each salt (Joyce,

Glaser, & Gerhardt, 2007). The two isomers of amphetamine act on different neural circuits, with

D-amphetamine improving circuits, which regulate hyperactivity, impulsivity, and sustained attention, and L-amphetamine acting only on circuits, which regulate sustained attention

(Sagvolden & Xu, 2008).

We selected Adderall

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for our experimental treatment due to its market prevalence and clinical efficacy. Adderall

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constitutes 25% of all prescriptions written for ADHD, and sales of its extended release form now surpass the sales of the main methylphenidate sustained release form, Concerta

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. Comparative studies have shown Adderall

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to be more effective in symptom regulation than atomoxetine (Sallee & Smirnoff, 2004). In addition, the combination of D- and

L-amphetamines is more potent than either isomer alone; the ratio in Adderall

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evokes a longer and higher volume dopamine response in striatum than either D-amphetamine alone or equal proportions of D- and L- amphetamines (Joyce et al, 2007).

Amphetamines inhibit the reuptake of certain neurotransmitters from postsynaptic space and increase their release, which increases neural firing. Thus, the proposed mechanism of action



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ADMINISTRATION IN FETAL NICOTINE RATS of Adderall

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via amphetamines is divided into two steps. First, it changes the conformation of

15 the dopamine transporter, which normally takes dopamine out of the postsynaptic space back into the presynaptic cell, such that the flow is reversed and dopamine flows out the of presynaptic cell after firing has ceased. Second, it inhibits the dopamine transporter, which prevents reuptake of the neurotransmitter into the presynaptic cell (Joyce et al., 2007).

Furthermore, it inhibits the activity of monoamine oxidases A and B (MAO-A and MAO-B), preventing it from breaking down the monoamines serotonin, dopamine, noradrenaline, and adrenaline, which stimulates their release after firing. The cumulative effect is that the available postsynaptic dopamine and noradrenaline is increased, augmenting the effect of firing by neurons which release these neurotransmitters, including those involved in the impulsive action neural circuit. This increase compensates for poor neurotransmitter modulation in this brain region in patients with ADHD (del Campo et al., 2011).

Methodology

Research Design

Our experimental design will have behavioral, neurological and pharmacological components. The independent variable for the behavioral component will be whether or not the mothers of the rats were administered nicotine during pregnancy; the corresponding dependent variable will be differences in SSRT and accuracy on stop trials. The neurological independent variable will be how neural firing in the dPL is influenced by both fetal nicotine exposure and

Adderall

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administration; the dependent variable for this aspect will be variations in neural firing of the dPL. Finally, for the pharmacological component, the independent variable is whether the fetal nicotine rats are administered with Adderall

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; the dependent variables are the changes in both behavior and neural activity between the three rat groups.



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Our experimental design will be divided into three experiments: a pilot study to examine the viability of fetal nicotine rats for this experiment, a drug administration experiment in which we will divide fetal nicotine rats into a drug experimental and a drug control group, and a fetal nicotine experiment in which we will compare unaltered Long-Evans rats to fetal nicotine rats.

Pilot Study

We will begin with a pilot study aimed to determine whether fetal nicotine rats can be trained to perform the stop-signal task. Unaltered Long-Evans rats have been successfully trained on this task before, but fetal nicotine rats may exhibit symptoms that prevent them from learning the task. We will obtain four male Long-Evans rats that have been exposed to nicotine during gestation from Charles River Laboratory. The exposure will be equivalent to human mothers smoking 2 to 3 packs of cigarettes per day during gestation. We will use males throughout the study because males are more even-tempered than females, and prenatal nicotine exposure has been shown to have more dramatic effects on males (Romero & Chen, 2004). The rats will be acclimated to human interaction and handling by introducing them to typical lab environments, such as the recording boxes. Once rats reach a subjective satisfactory level of competence on this task, we will begin training on the stop-signal task.

The stop-signal task will be conducted in aluminum boxes equipped with fluid wells and directional lights. House lights will also be located above the wells and signal lights.

Furthermore, task control will be implemented via computer; port entry and reward retrieval are monitored by photobeams. First, the rats will learn to associate light with reward direction (left light leads to reward in left well). These trials will be called go trials. Once they have been able to maintain an acceptable performance of 70% correct, stop-signal trials will be added. A minority of the trials will have a second stimulus dictating the inhibition of the first light flash



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17 and require a switch to the other well to receive the reward. A pseudo-random sequence in which left and right trials are mostly random will be employed. This sequence ensures that the ratio of right and left occurrences remains 1:1. There cannot be more than three of the same direction trials in a row. This ensures equal sampling of all trials during the course of a session.

After a month of daily stop-signal sessions, we will evaluate the performance of the rats.

First, we will look at the differences between the percentages of correct stop trials and go trials.

If we see a significant difference between these percentages, then we can conclude that the stopsignal task is effectively measuring the extent of impulsivity of the rat. We will also look at the

SSRT to obtain an empirical measure of impulsivity in the rats.

The completion of the pilot study will allow us to conclude whether fetal nicotine rats are capable of completing the stop-signal task. If we determine that the rats are incapable of completing the task, we will investigate other models, such as the SHR.

Adderall

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Study

In this section of our study, we will begin by constructing the electrodes for neural recordings. After the electrodes have been constructed and determined to be functional (see

Appendix C), sixteen fetal nicotine rats will be obtained and trained on the stop-signal task. Once training is completed, we will perform the surgeries to implant the electrodes. To begin, rats will be anesthetized with isoflurane, and fixed within ear bars to ensure stability throughout the surgery. An incision will then be made to insert the electrode. The incision will then be stapled together, and the rat will be administered buprenorphine and placed into a recovery chamber.

There will be experts in the laboratory who will be able to ensure that all aspects of surgery, especially accurate electrode placement, are performed correctly.



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The rats will need to recover post operatively for one to two weeks (Bari et al., 2011;

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Acheson et al., 2006). Buprenorphine will be administered twice during the 24-hour period following surgery for acute pain relief. Cephalaxin 15mg/kg will be administered postoperatively twice daily for two weeks to prevent bacterial infections.

Each rat will perform 240 trials in one session over two hours per day, five days per week. Given the available laboratory equipment and number of researchers who are competent in performing the methodology, this design is highly feasible within the time constraints of one semester. The electrode is advanced 4µm following each session. Neural firing will be recorded by electrodes inserted alongside single cells in the dPL; a frequency distribution of action potentials during trials will be plotted as a function of time. If these plots of neural firing show an increase in activity only in response to the stop-signal during correct ‘stop’ trials, this will allow us to establish that the brain region is active during response inhibition rather than in anticipation of a reward or in response to the ‘go’ signal, thus correlating the dPL with regulation of impulsivity. Observation of task performance will help us compare and contrast typical

ADHD behaviors associated with rat models in this study and those in humans (Calu, Roesch,

Haney, Holland, & Schoenbaum, 2010).

Eight of the rats will be chosen through matched random selection to receive Adderall

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Administration of the drug will be through systematic injections that are given subcutaneously.

Rats assigned to the drug control group will receive injections of saline to eliminate potential confounding variables caused by injections. Each rat will be injected with a dose of 1 ml/kg body weight of the animal. Drugs will be administered every three days. All dosages will be giving



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ADMINISTRATION IN FETAL NICOTINE RATS according to a balanced procedure (Sagvolden & Xu, 2008). We will obtain the Adderall

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from

Henry Schein, a worldwide pharmaceutical distributor.

Neural Recordings

As the rats complete trials, we will conduct single-unit recording. Neural activity will be monitored using a computer interface in the chambers (Bryden, Johnson, Diao & Roesch, 2011).

We will screen the electrode wires prior to each trial to determine whether activity is detected. If the electrodes are unable to detect neural firing, we will advance the electrode by 80µm. If activity is detected, then the trial will be recorded; following the session, the electrode will be advanced. The electrodes detect action potential waveforms. During recordings the signals detected by the electrode will be amplified, and then be filtered in order to identify and save specific action potential waveforms (Roesch, Calu, & Schoenbaum, 2007).

Fetal Nicotine Study

In this portion of our study, we will obtain eight male Long-Evans rats that do not have any genetic modifications or environmental interferences as well as eight male fetal nicotine rats.

These rats will undergo the same stop-signal training and surgery outlined above and will receive saline injections like the drug control group. They will also be subject to the same neural recording processes. Unaltered Long-Evans rats serve as a control to the fetal nicotine rats because they do not have the model of ADHD. Because of this condition, these rats will serve as a baseline for behavior on the stop-signal task and neural firing in the dPL.

Data Analysis

Data will be analyzed in MATLAB to obtain firing and behavioral information.

Wilcoxon tests, t-tests, ANOVA, and Pearson Chi-square tests will be implemented to compare and measure relevant statistics (Bryden et al., 2011). Examples of analyses include comparative



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ADMINISTRATION IN FETAL NICOTINE RATS histograms of neural firing patterns across the trial time course as well as observing the

20 relationship between SSRT and neural firing. When a rat’s session is analyzed, the intensity and timing of its neural firing is compiled and aggregated with other sessions to prove an informative comparison of neural activity of the dPL in all groups of rats.

Histology

Histological analysis will be performed to confirm that the electrodes were placed in the correct region of the brain during surgery. The analysis is divided into three major components: brain sectioning (slicing the brain into thin layers), placing the sections on slides and applying a cresyl violet stain, and comparing the sections to a brain atlas (see Appendix D). A microtome will be used to accurately and efficiently section the brain tissue into micrometer thick fractions.

The unstained parts of the tissue will be coated with a clearing agent (Barth, 1997).

Animal Care

We will submit a proposal of our study for review by University of Maryland

Institutional Animal Care and Use Committee. Throughout the study, we will adhere to the procedures outlined in the Guide for the Care and Use of Laboratory Animals (Garber et al.,

2011) . Using these guidelines, we will house our rats in appropriate cages with proper room temperature, ventilation, and feeding. When euthanizing the rats, we will use isoflurane to place them in a deep unconscious state. Following this, we will perfuse the rats by introducing a needle to the circulatory system of the rat through the heart. Saline and a fixative will be pumped through the circulatory system to preserve the brain tissue. The rats will then be sacrificed by decapitation. The brains will be excised and then stored in a refrigerator.

Study Limitations



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A main concern for our study will be that it may suffer from effects of attrition. Factors

21 such as fatigue, hunger, and thirst will alter the rats’ motivation levels, which will force us to disregard trials that were adversely affected by these conditions (Prescott et al., 2010); that is, trials during which the rat did not complete the entire session. We will be able to control for these variables by ensuring that the rats will not be subjected to exhaustive tests and that they will be allowed ample rest time between trials. In order to ensure that trials are executed efficiently, we will mildly deprive rats of water prior to completing the trials and use a thirstbased reward system. The rats will receive 35 mL of water per day. Several hours prior to running the task, the rats will not receive water. This lack of water will act as an incentive to motivate the rats to perform the task in order to receive water as a reward.

It is also necessary that the rats undergo a full recovery prior to performing the stopsignal task by allotting recovery time after surgery. An insufficient maturation period poses a medical risk to the rats and places the rats under additional stress that may alter the results of the stop-signal task (Krishnan, Panigrahi, Jayalakhsmi, & Varma, 2011). On the contrary, neuroplasticity may occur if the rats are given too much recovery time. A one to two week recovery period should be sufficient to avoid both stress and plasticity.

There will also be a possibility of experimenter error in our study. A small group of team members will be given the opportunity to build the electrodes and implant them into the rats’ brains. If the building or implanting of the electrode differs between members, this may affect the validity of our results. To compensate for any differences, we will follow a set of consistent procedures. In addition, post-mortem histology will reveal whether or not electrode placement was correct; we will not include data collected from incorrectly placed electrodes in our final data analysis.



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22

In order to account for a possible influence of age on impulsivity, we will only use male rats of the same age. We will obtain our rats from Charles River Laboratory, which will breed the fetal nicotine rats by following an established process for exposing rats to pharmaceuticals during pregnancy. By accounting for these variables, we will preserve the internal validity of our research.

Conclusion

The significant increase in the number of ADHD diagnoses made in recent years can be attributed to the lack of an experimentally and clinically verified neurological basis of ADHD.

The criteria for diagnosing the disorder as provided by the DSM-IV allows for a diagnosis to be made primarily from qualitative observations of a patient’s behavior. The high incidences of misdiagnoses, however, have shown that such observations are insufficient in generating a proper diagnosis. The association of ADHD to a particular brain region would be of great value to health professionals and pharmaceutical companies by delivering drug treatments to specific regions of the brain involved in monitoring ADHD symptoms.

Previous research has indicated that the dPL is involved in controlling impulsivity, one of the most common symptoms of ADHD. Other studies have shown that in an individual with

ADHD, there is a reduction in the available quantity of the neurotransmitters dopamine and noradrenaline. This effect can be counteracted by Adderall

®

, one of the most widely prescribed medications for ADHD treatment. This amphetamine-based drug has been shown to increase the amount of dopamine and noradrenaline in the dPL cortex and, consequentially, reduce impulsivity in both rat models of ADHD. We aim to prove that the physiological pathways within the dPL are disrupted in their inhibition of impulsivity by conducting single-unit recordings of fetal nicotine rats and comparing these to recordings of Long-Evans rats. In



®

ADMINISTRATION IN FETAL NICOTINE RATS addition, with the administration of Adderall

®

, we expect that these effects should be reversed,

23 which will be evident by a change in the single-unit recordings following administration in the fetal nicotine rats.

In order to test the effects of Adderall

®

, we will determine the validity of the fetal nicotine rat as a model of ADHD by establishing that impulsivity caused by fetal nicotine exposure is a symptom of ADHD. Through the stop-signal task, we expect to see an increase in

SSRT values in the rats, which is a measure of increased impulsivity. In addition, in the singleunit recording studies conducted during the task, we expect to see decreased dPL activity. These results would suggest that neural firing in the dPL is correlated with impulsivity, and that

Adderall

®

administration alters neural firing patterns within the dPL. The findings obtained from our research can be applied to humans because the data will be recorded from a homologous brain area between humans and rats. Even if our results do not support our hypothesis, our findings will contribute to further research using rat models and neuron firing recordings.



®


Appendix A: Anticipated Budget

Item Total cost ($)

Plexon Recording System

Cost per unit

($)

~70,000

Amount of item

1

Test boxes equipped with photobeams

Electrodes

Adderall

®

Long-Evans rats

Fetal Nicotine rat mothers*

~15,000

26.45

~110

~100

~500

8

8

4

24

1

Provided by mentor


211.60

440

2400

500

24

Cables ~800 8 Provided by mentor

Histology supplies (saline, stains, etc.)

~2000

Surgical supplies (anesthetics, etc.) ~15000

N/A

N/A



Total: $3551.60

* We will be purchasing the rats after gestation, but our supplier is in the process of making an estimate. Four mothers would be enough to obtain sixteen male rats.



®


Appendix B: Timeline for Success

Spring 2013



Stop-signal task and single unit neuron recording with control group

25

Spring 2012



Finalize and present thesis proposal



Apply for IACUC approval



Apply for grants: HHMI, NIDA



Construct Schoenbaum electrodes



Create team website

Summer 2012



Pilot study to determine if fetal nicotine rats will be able to successfully perform the stopsignal task



Obtain Adderall

®

for study

Fall 2012



Implant electrodes and run rats through stop-signal task and single unit neuron recording with experimental groups one and two: (1) fetal nicotine rats with saline and (2) fetal nicotine rats with Adderall

®



Outline thesis chapters



Draft thesis chapters 1 and 2



Present preliminary findings at Junior Colloquia



Continue updating team website, consulting with librarian for editing thesis paper, and searching for conferences to attend



®




Surgery with control group (Long-Evans) and fetal nicotine drug control group, stop-

26 signal task retest



Histology studies with Nissl staining for experimental and control groups to verify electrode location



Present preliminary findings at Undergraduate Research Day



Revise team thesis paper chapters 1-3 based on feedback from librarian and mentor



Continue updating team website and searching for conferences to attend

Fall 2013



Data analysis with ANOVA, MATLAB, t-tests, Wilcoxon tests, and chi-squared tests



Complete thesis paper draft



Attend senior orientation in September



Prepare for the Team Thesis Conference rehearsal in February



Contact and determine discussants for thesis presentation



Continue updating team website, consulting with librarian for editing thesis paper, and searching for conferences to attend

Spring 2014



Practice presentation at rehearsal



Complete thesis paper



Present findings at Team Thesis Conference



Submit final thesis



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Appendix C: Schoenbaum Electrode Construction

Materials needed to construct an electrode:

 Nichrome/Formvar wire: 0.0010” bare, 0.0015” coated



Cannula with 27 gauge thin wall diameter



Auguts: both intact and pins from auguts that have been pushed out



Soldering wire



Super glue



Flux (cleaning, purifying, and flowing agent used to aid in metal joining)



Measuring calipers



Silver paint



Forceps, both blunt and sharp



Scissors



Constructed augut holder



Battery



Two alligator clip wires



Saline solution and small beaker



Reamers (metalworking tools used to create precise holes)



Permanent marker

Construction of parts:

1.

Push augut pins out by hand using a cannula piece.

2.

Take cannula of 0.4 mm diameter and measure with calipers to 15 mm length and make sure to bevel the edge of the cannula and clean out the hole with a reamer.

3.

Cut off the narrow end of the augut pin.

27



®

28


4.

Place cut down augut pin into a tightened holder and solder, using flux, the cannula to the augut pin attaching the beveled end to the pin with the beveled side facing up. The cannula should be straight and centered on the augut pin in all dimensions.

Construction of the electrode:

1.

Cut 11 pieces of wire at 8cm long.

2.

Gather wires and roll all wires together to create a tight bundle.

3.

Wet the ends of the bundle that will be sent through the cannula for ease.

4.

Feed the wires into the cannula and cut off the tip that was moistened.

5.

Bend the other end of the wires (near the beveled edge of the cannula) up and place a small drop of flux and super glue where the wires come out of the cannula.

6.

Wait for wires to dry and then carefully place the cannula on the middle pin of the augut that is being held by a constructed augut holder.

Under a microscope:

1.

Wrap the wires around the pins with forceps with each individual wire wrapped around each pin 2-3 times. Strip the ends of the wires for better conductivity. Pull the remaining wire with the forceps as close to the pin as possible and cut it right next to the pin. Wires to the left should be wrapped clockwise and wires to the right should be wrapped counter-clockwise.

2.

Paint the wire and pin with silver paint. Place a droplet on top of an insect pin and place this droplet on top of the pin and gently and slowly pull the droplet down the pin to cover the wire completely and to ensure that the tip of the wire is in contact with the pin.

3.

Let the first coat dry and then repaint the same way.



®

29


4.

Check conductivity after both coats have dried by placing wire tips into saline solution in a small beaker. Then place a one side of a two-sided alligator wire onto a battery and the other to the beaker of saline. With another wire, connect one side to an insect pin to touch the flip side of the augut for each of the pins and the other side to the other part of the battery. Check for bubbles for each wire and if no bubbles are seen then look for a loose end to repaint or strip the paint with a razor blade and start over.

To implant the electrode, holes will be drilled in predetermined positions on the rat’s skull for anchoring screws that will hold the electrode in place, and a somewhat larger central hole will be made for insertion of the electrode itself. With a microscope, the outermost layers of the membranes that cover the brain will be cut away from this central hole and the microelectrode will be inserted into the brain tissue. The electrode will be inserted further into the brain at a rate of 100 microns/minute until it is near a single neuron so that recording can begin. The electrode will then be screwed to the skull and fastened using grip cement and dental acrylic.



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Appendix D: Histology with Nissl Staining

In order to ensure that the electrodes in the study were, in fact, recording from the dPL

30 we will perform histological analysis. Histology is the study of animal tissue under a microscope, as well as the techniques that prepare the tissue for microscope study. To be viewed by light microscopy, tissue must be stained in order to trace fiber tracts and receptor types. First, brain tissue must be isolated by sacrificing the animal of interest and then perfusing it to drain blood.

Blood may interfere with the staining process so instead a fixative is added into the vascular system, which also helps harden the brain. The brain is then removed, sectioned into thin slices, and stained with cresyl violet. Once it is stained, structures can be identified using an atlas of the rat brain.

Materials need for histology:



Fixed rat brain



Cryostat or microtome to section the brain accurately



Microscope slides



For the stain solution (1.5 L of 0.25% thionin): o 1428 mL distilled water o 54 mL 0.1M sodium hydroxide (NaOH) o 18 mL glacial acetic acid o 3.75 g thionin

To create stain:

1.

Mix distilled water, sodium hydroxide, and acetic acid. Heat until just boiling, then add thionin and reflux for 45 minutes with stirring.



®


2.

Cool to room temperature, then decant 1000 mL of the solution in a dark bottle. Decant

31 the remainder of the solution into another dark bottle and store this excess.

3.

Keep stain at 37 degrees Celsius. Filter solids out before each use.

To perform a Nissl stain:

1.

Mount dried, sliced tissue on slides.

2.

Defat the tissue in a fume hood in a solution of equal parts concentrated chloroform and ethanol for one hour.

3.

Soak tissue in 100% ethanol twice for two minutes at a time, followed by 95% ethanol,

70% ethanol, and 50% ethanol each for two minutes at a time. Dip twice in distilled water twice.

4.

To stain, soak tissue for 20 seconds in 0.25% thionin, followed by two dips in distilled water twice.

Dip in 50% ethanol, 70% ethanol, 95% ethanol twice, and 100% ethanol twice for 4 minutes each, followed by 4 minute soaks in ortho-dimethylbenzene, meta-dimethyl benzene, and paradimethylbenzene. Let dry thoroughly.



®


Appendix E: Glossary of Terms

Action potential

A neuron contains a plasma membrane with a voltage differential caused by ion pumps and ion channels. When a neuron is at rest, it has a resting potential of -70 millivolts. When it

32 receives electrical signals from dendrites of other neurons to its axon, it reaches a threshold potential of -55 millivolts. At this point, an action potential occurs and the membrane potential shoots up to around +100 millivolts. This potential travels to dendrites of the neuron and is passed on to other axons.

Amphetamines

Amphetamines, from alpha-methylphenethylamine, are psychostimulant drugs which increase alertness and focus. This class of drugs works by modulating the dopaminergic and noradrenergic neurotransmitters in specific region of the brain. Modulation is achieved by altering the DA reuptake protein, preventing the reuptake of DA.

Animal/Rat models

Animal models are used to represent various diseases and are evaluated based on three criteria: face validity, construct validity, and predictive validity. Face validity is the model’s ability to display major symptoms of the disease. Similarities to the disease’s pathophysiology exemplify construct validity. Predictive validity is present when the model responds in a similar fashion to drugs designed to alleviate the disease. An excellent animal model for a disease will display all three forms of validity.

Augut

The augut is a connector crimp tool that allows for smooth cable connections during usage. This is mainly used for electrode construction.



®


Cannula

33

A small flexible tube placed into a body cavity or blood vessel, which is used to insert a surgical instrument, drain off fluid, or administer medication. For our research, we will surgically implant the cannulas into the rats to deliver the electrodes to the dPL.

Dorsal prelimbic cortex

The dorsal prelimbic (dPL) cortex is located within the PFC, the brain region that serves as the primary control for complex cognitive behaviors. Because this area controls higher-level executive functions, it stands to reason that the dPL would be involved in controlling impulsivity; this assumption has been supported by various studies. Lesioning the dPL has been shown to impair decision-making involving information regarding actions the subject is about to perform; hence, inactivation of the dPL has also been shown to increase stop-signal reaction times (Bari et al., 2011).

Impulsive action

In the stop-signal task, impulsive action can be observed when the rats fail to change direction or when a premature response occurs. Essentially, impulsive action happens when there is no behavioral inhibition. Impulsive action can be distinguished from impulsive choice, which occurs when decisions are made without any consideration. Rats exhibit impulsive choice when they choose a smaller immediate reward as opposed to a larger delayed reward.

Impulsive action neural circuit

When recording the subject’s neural activity, it is important to remember that neural signals are rarely a lone signal. Rather, they tend to take complex pathways through various brain regions to achieve the desired physical output. For impulsive action, this circuit includes the PFC, orbitofrontal cortex, anterior cingulated cortex, and nucleus accumbens. Therefore,



®

ADMINISTRATION IN FETAL NICOTINE RATS when a drug is administered to a single region of the brain it will only affect that area, not the

34 entire circuit in which the signal is traveling. For this study, we will be administering drugs systematically, which will affect the entire brain, not just the dPL.

Neural circuit

The neural circuit represents multiple brain regions through which a neural signal passes through to execute a physical action.

Neuroplasticity

Also known as brain plasticity, neuroplasticity is the ability of the brain to reorganize existing connections between neurons. When a particular brain region is damaged, the intact neurons of functional brain regions are able to grow new nerve endings to connect to damaged brain cells or to other functional neurons to form a new network of signaling. In this way, the brain is still able to perform necessary functions even if the regions normally responsible for performing these functions are injured.

Neurotransmitters

A neurotransmitter is a chemical that transmits signals from a neuron to another cell via a synapse, the structure between a neuron and another cell. A neurotransmitter leaves the presynaptic neuron, crosses the synapse, and arrives in the postsynaptic neuron. Noradrenaline

(NA), also known as norepinephrine, is a neurotransmitter that commonly influences alertness and one’s reward system. Dopamine (DA), another neurotransmitter, is a precursor to norepinephrine in the biosynthesis, involved in cognitive functions including attention. Reuptake is the process in which a neurotransmitter, such as DA or NA, is reabsorbed into a neuron after it has transmitted the neural impulse. Neurotransmitters can also be degraded extracellularly by acetylcholinesterase. Due to the large molecular size of neurotransmitters, reuptake can only be



®

ADMINISTRATION IN FETAL NICOTINE RATS achieved with specific transporter proteins that carry the molecules across the cellular membrane.

Nissl Stain

The nissl stain is a histological procedure used to identify differences in neurons and

35 therefore differences in brain areas. The stain colors the RNA molecules (most likely ribosomal

RNA (rRNA) blue, which are in the cytoplasm of the cell at the neuron’s time of death. The stain stains the RNA molecules (most likely ribosomal RNA (rRNA)) that are in the cytoplasm of the cell at the time of the death of the neuron blue. This stain shows RNA molecules and the rough endoplasmic reticulum, an organelle in the cell, which modifies proteins to make them functional for the cell to use, has rRNA on the surface of the organelle. The stain shows the structural features of the neurons. After reviewing the stains, we will use a brain atlas to identify the brain region we are in.

Prefrontal cortex

The prefrontal cortex (PFC) is part of the frontostriatal circuit, which is known to control higher-level executive functions, including inhibition. Within the PFC is the dorsal prelimbic area, the primary region of study in this investigation.

Single-unit neural recording

Single-unit neural recordings will be procured by implanting a Schoenbaum electrode into the subjects’ dorsal prelimbic area of their prefrontal cortex. These electrodes take extracellular recordings of the changes in electrical potential, thereby measuring when the neuron fires, indicating neural activity.

Stop-signal task



®


The stop-signal task is a behavioral measure of impulsivity. Subjects are first trained on

36 the task, learning how to respond correctly to the visual input (e.g. right light ⇒ go right). When the task begins, subjects are directed to go to the right or left well and, if they choose the correct side, they are rewarded with a quantity of water with 10% sucrose. The time it takes for the subject to complete this task is defined as the “go reaction time.” This sequence makes up a majority (80%) of trials, so the subjects become accustomed to going to the same side the light flashes. However, during the other 20% of trials, a primary light will flash, directing the subject to one side, while the subject in en route to the directed well, the opposite side’s light flashes, instructing the subject to change direction. The time it takes the subject to correct their direction is defined as the “stop reaction time.” If the subject does not change direction, this is considered an incorrect response (see Figure 2).

The dependent variable of the stop-signal task is the time it takes the subjects to correct their response after the second light flashes, which is affected by the independent variable, the time between the primary flash and the secondary flash. These times are measured by photosensors across the well that, when broken, record the difference in time.

Figure 2. Diagram of the stop-signal task (Eagle & Baunez, 2010).

The levers shown in this diagram would be wells in our experiment.



®


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