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Running Head: Validating an Animal Model of ADHD
APA 6th
Validating an animal model of attention deficit hyperactivity disorder: neural correlates of
impulsivity in prefrontal cortex mediated by Adderall® in fetal nicotine rats
Team Research in Testing ADHD’s Link to Impulsivity 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
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Table of Contents
Abstract ...........................................................................................................................................3
Introduction ....................................................................................................................................4
Research Questions & Hypotheses ..............................................................................................6
Implications ..................................................................................................................................6
Literature Review ..........................................................................................................................7
Clinical Components ....................................................................................................................8
Fetal Nicotine Rat Model .............................................................................................................9
Stop-Signal Task ........................................................................................................................10
Neurophysiology ........................................................................................................................11
Pharmacology .............................................................................................................................12
Methodology .................................................................................................................................13
Research Design .........................................................................................................................13
Pilot Study ..................................................................................................................................14
Adderall® Study..........................................................................................................................15
Neural Recordings ......................................................................................................................16
Fetal Nicotine Study ...................................................................................................................17
Data Analysis .............................................................................................................................17
Histology ....................................................................................................................................18
Animal Care ...............................................................................................................................18
Study Limitations .......................................................................................................................18
Conclusion ....................................................................................................................................19
Appendix A: Anticipated Budget ...............................................................................................22
Appendix B: Timeline for Team Success ...................................................................................23
Appendix C: Electrode Construction .........................................................................................24
Appendix D: Histology with Nissl Staining ...............................................................................27
Appendix E: Glossary of Terms .................................................................................................30
Appendix F: Figures ....................................................................................................................35
References .....................................................................................................................................37
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Abstract
Currently, the neurological basis of attention deficit hyperactivity disorder (ADHD) is not well
established; this disorder is diagnosed qualitatively using behavioral tests 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 performance of a stop-signal task. We will also establish whether Adderall®
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 an animal model of attention deficit hyperactivity disorder: neural correlates of
impulsivity in prefrontal cortex mediated by Adderall® 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, last through adulthood. Children with
ADHD are more likely to encounter academic difficulties, such as scoring poorly on exams and
withdrawing prematurely from school (Karande & Kulkarni, 2005). Recent research predicts that
ADHD affects approximately five to ten percent of school aged children (Evans, Morrill, &
Parente, 2011).
This disorder has caused controversy due to 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, thus the disorder has been diagnosed
based on subjective, behavioral observations rather than reliable objective identifiers of the
disorder. This ineffective method of diagnosing ADHD has led to numerous misdiagnoses, rising
medical costs, and over-prescribed 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 patient 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
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). Poor performance on these tasks is observed after
pharmacological manipulation of prefrontal cortex, notably the dPL, suggesting an association of
the prefrontal cortex with response inhibition (Aron & Poldrack, 2004; Bari et al., 2011).
Unfortunately, no single neuron recording study has been performed in conjunction with
performance on the stop-signal task, so it is still unclear what dPL signals during impulse control
in normal animals or in animal models of ADHD.
Numerous animal models have been suggested for ADHD, but 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). Our
study will serve to confirm or disprove the fetal nicotine rat as a plausible model of ADHD by
demonstrating behavioral deficits during performance of a stop-signal task, showing that taskrelated activity in dPL is disrupted during stop-signal task performance, and that both behavioral
and neural deficits are corrected with administration of Adderall®.
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 administering Adderall®, a mixed amphetamine salt that has been
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shown to reduce ADHD symptoms in patients (Weisler, 2005). The impact of Adderall® on
neurotransmitters has been established through previous research; 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 accomplished by linking Adderall® 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 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® 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 that fetal
nicotine rats will show increased impulsivity by having reduced neural firing in the dPL in
response to the stop-signal. Finally, we hypothesize that Adderall® administration will increase
dPL neural firing and decrease stop-signal performance in rats that exhibit increased impulsivity.
In recent years, the dramatic increase in the number of ADHD diagnoses can be
attributed to generally qualitative observations of an individual’s behavior; the lack of a
clinically significant and verified neurological basis has resulted in significant increases 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 aid in the formulation of a
concrete diagnosis of the disorder, which may reduce incidences of misdiagnosis.
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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 homologous to structures in the human prefrontal cortex (Quirk & Beer,
2006). The prefrontal cortex has been shown to be disrupted in ADHD patients (Aron, Fletcher,
Bullmore, Sahakian, & Robbins, 2003). After analyzing the association of the dPL and
impulsivity, we will determine whether Adderall® administration increases neural firing in the
dPL. This neural activity is associated with improved task performance in response to the stopsignal, and will elucidate the causal relationship between alterations in 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 more effective treatments that target dPL and areas connected to it. Further, the
precise resolution of single neuron recordings will allow us to pinpoint the signals involved in
impulsive action, allowing drugs to target those signals and not others that might lead to
unwanted side effects.
Literature Review
The first step in establishing the neural basis of ADHD is to select a rat model and verify
its validity through the use of neural recordings and pharmacological administration. In order to
find a neural basis of ADHD, we must choose a valid rat model of the disorder and integrate it
with Adderall® administration in addition to neural recording of the dPL. We will begin with an
overview of the clinical components of ADHD to assess the deficiencies in the current system of
diagnosis. We will analyze the fetal nicotine rat model, which has been shown to represent
ADHD symptoms but requires further study to validate it as an accurate model of ADHD. We
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will then examine relevant research on the neurophysiology of ADHD, specifically that which
concerns the dPL and the neurotransmitters dopamine and noradrenaline. 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 (Oosterlaan, Logan, & Sergeant, 1998). The stop-signal
task is a strong measure of both inhibition and impulsivity. Finally, we will examine Adderall®, a
common treatment for ADHD that inhibits dopamine and noradrenaline reuptake, which then
reduces impulsivity (DuPaul & White, 2006). To the best of our knowledge, there has not been a
study of neural recording during stop-signal performance while under the influence of Adderall®.
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
the manual, the most common symptoms associated with the disorder are impulsivity,
inattention, and hyperactivity (American Psychiatric Association, 1994). 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
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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.
Fetal Nicotine Rat Model
The fetal nicotine rat model serves to represent various ADHD symptoms and brain
deficiencies similar to those found in humans. Fetal nicotine rats are those 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 state 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, an effective animal model should demonstrate three core
symptoms of ADHD to be present: attention deficit, hyperactivity, and impulsivity. Sontag et al.
also found that construct validity shows that the model corresponds to an established
pathophysiological basis of the disorder. 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. Since 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 are many different
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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 problems during fetal development of brain areas such as the
prefrontal cortex that are involved in inhibition of actions 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. These behavioral symptoms are also
present in the fetal nicotine model, and thus demonstrate face validity. Finally, it is worth noting
that, even if this rat model does not prove to be a valid model of 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 (Logan, 1994). The task gauges how quickly an initiated response is inhibited (Eagle
& Baunez, 2010). In the task, the participant is trained to respond to a conditioned stimulus such
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as a light turning on. On a minority of trials, another stimulus will be presented after the first one
and serves as a signal to stop the initial response. We will use the stop-signal task because it
provides a quantitative measure of motor inhibition by examining the stop signal reaction time
(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 (van Boxtel, van der Molen, Jennings, & Brunia, 2001). ADHD patients
exhibit 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
The prelimbic cortex is a subsection of the prefrontal cortex in both human and rat brains
that functions as an executive control center. 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).
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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
spontaneous, unpredictable 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 ADHD drugs such as atomoxetine on improving SSRTs. When
atomoxetine is used to locally block the noradrenaline reuptake in the dPL, stop-signal task
performance in SSRTs improves. Since prelimbic cortex lesions affects performance on
behavioral tasks like these, it is thought to be involved in action selection, planning, inhibition
and attention. Deficiency in these functions could result in poor performance on the stop-signal
task making dPL a strong choice for study. Further, it has been implicated in impulsivity and
ADHD and is present in both rats and humans, yet it is unclear how neural firing in the dPL
might change as a result of exposure to prenatal nicotine or Adderall®.
Pharmacology
Various pharmacological treatments in addition to behavioral therapies are administered
to patients to manage ADHD symptoms. The three main chemical components of ADHD
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pharmacological treatments are atomoxetine, methylphenidate, and amphetamine. The most
common treatment for ADHD in patients is amphetamine stimulant administration, with the
mixed amphetamine salts in Adderall® being the most effective. Amphetamines have been
shown to effectively reduce hyperactivity, impulsivity, and inattention in patients with ADHD
(Weisler, 2005).
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® 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®; the dependent variables are the changes in
both behavior and neural activity between the three rat groups.
Our experimental design will be divided into three experiments: a pilot study to examine
the viability of fetal nicotine rats for this experiment by breeding fetal nicotine rats, an Adderall®
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.
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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 ten male and twenty female Long-Evans rats from Charles River
Laboratory. Fetal nicotine rats will be bred using nicotine-infused water dosages. 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). Once rats reach a subjective satisfactory level of competence on this training
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
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.
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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 based on statistical t-test analysis between these percentages,
then we can calculate an SSRT to measure the extent of impulsivity of the rat.
The completion of the pilot study will allow us to conclude whether fetal nicotine rats are
capable of performing accurately on the stop-signal task. If we determine that the rats are
incapable of completing the task, or if we do not observe a significant between-group difference,
we will investigate other models, such as the spontaneously hyperactive rat (SHR), or try using
the delay discounting task.
Adderall® Study
In this section of our study, we will begin by constructing the electrodes for neural
recordings. After the electrodes have been 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, the skull will be drilled, and the electrode will be inserted. The
incision will then be stapled together, and the rat will be administered buprenorphine and placed
into a recovery chamber. Our mentor will be able to ensure that all aspects of surgery, especially
accurate electrode placement, are performed correctly.
The rats will need to recover postoperatively for one to two weeks (Bari et al., 2011;
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.
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After the recovery period, each rat will perform 240 trials in one two hour session 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 will be advanced 40 µm following each session.
Neural firing will be recorded by electrodes inserted alongside single neural cells in the dPL; a
frequency distribution of action potentials across neural membranes 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 on stop signal
performance to receive Adderall® throughout the duration of the study. The drug will be
administrated orally in chocolate milk through a syringe. Rats assigned to the drug control group
will receive chocolate milk only. 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 given according to a
balanced procedure (Sagvolden & Xu, 2008). We will obtain the Adderall® 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
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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. 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 control 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
chocolate milk through a syringe 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 serving 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
histograms of neural firing patterns across the trial time course as well as observing the
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.
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Histology
Histological analysis will be performed to confirm that the electrodes were placed in the
correct region of the brain during surgery. See Appendix D for details (Barth, 1997).
Animal Care
We have submitted 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
Attrition effects are a main concern for our study. Factors 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; that is, trials during which the rat did not complete the
entire session (Prescott et al., 2010). 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 thirst-based reward system. The rats will
receive 35 mL of water per day. Several hours prior to running the task, the rats will not receive
VALIDATING AN ANIMAL MODEL OF ADHD
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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 recovery 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).
There will also be a possibility of experimenter error in our study. A small group of team
members will 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 and electrodes
and surgeries will be divided evenly between control and experimental groups. In addition, postmortem 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.
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, and we will then
breed the rats by following an established process for exposing rats to the 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 is primarily composed of
qualitative observations of a patient’s behavior. The high incidences of misdiagnoses, however,
VALIDATING AN ANIMAL MODEL OF ADHD
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have shown that such observations are insufficient in generating a proper diagnosis. The
association of ADHD to a particular brain signal would be of great value to health professionals
and pharmaceutical companies by delivering drug treatments to specific regions of the brain that
modulates such a signal and would ultimately be critical for treating ADHD symptoms without
unwanted side effects.
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®, a common medication 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 signal within the dPL are disrupted in rats
with high impulsivity by conducting single-unit recordings in fetal nicotine rats and comparing
these to recordings of controls. In addition, with the administration of Adderall®, we expect that
these effects should be reversed, 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
VALIDATING AN ANIMAL MODEL OF ADHD
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 will not support our hypothesis, our
findings will contribute to further research in that we will determine the contribution of dPF to
impulsive behavior in normal rats and if this signal is disrupted in prenatally nicotine exposed
rats.
21
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Appendix A: Anticipated Budget
Item
Cost per unit
($)
Amount of
item
Total cost ($)
Plexon Recording System
~70,000
1
Provided by
mentor
Test boxes equipped with
photobeams
~15,000
8
Provided by
mentor
Long-Evans rats
27
10
270
Animal Care
~200/month
6
1200
Electrodes
~100
24
2400
Adderall®
~500
1
500
Cables
~800
8
Provided by
mentor
Histology supplies (saline, stains,
etc.)
~2000
N/A
Provided by
mentor
N/A
Provided by
mentor
Total:
$4370.60
Surgical supplies (anesthetics, etc.) ~15000
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Appendix B: Timeline for Success
Spring 2012

Finalize and present thesis proposal

Apply for IACUC approval

Apply for grants: HHMI, NIDA

Construct 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 and control group: (1) fetal nicotine rats with saline. (2) fetal
nicotine rats with Adderall® and (3) Long-evans rats.

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
Spring 2013
VALIDATING AN ANIMAL MODEL OF ADHD

Stop-signal task and single unit neuron recordings if more rats are needed. 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
24
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Appendix C: 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.
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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.
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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
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.
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2. Cool to room temperature, then decant 1000 mL of the solution in a dark bottle. Decant
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.
VALIDATING AN ANIMAL MODEL OF ADHD
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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
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.
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Cannula
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,
VALIDATING AN ANIMAL MODEL OF ADHD
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when a drug is administered to a single region of the brain it will only affect that area, not the
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
VALIDATING AN ANIMAL MODEL OF ADHD
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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
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.
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Stop-signal task
The stop-signal task is a behavioral measure of impulsivity. Subjects are first trained on
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.
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Appendix F: Figures
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).
VALIDATING AN ANIMAL MODEL OF ADHD
Figure 2. Diagram of the stop-signal task (Eagle & Baunez, 2010). The levers shown in this
diagram would be wells in our experiment.
36
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