Neoclomipramine Rat Model Validation

Running head: NEOCLOMIPRAMINE RAT MODEL VALIDATION 1
Evaluation of the Validity of a Neoclomipramine Rat Model for Obsessive-Compulsive Disorder
Using the Hole Poke Board and Marble Arena Behavioral Assays
Kirstin Craig
Colgate University
May, 2014
Acknowledgements
I’d like to thank Professor Deborah Kreiss for her help and advice throughout this process, as
well as for providing me with the approved animal study ready to go at the beginning of the fall
semester. Thank you to Professor Albert for the help with data analysis, as well as Professor
Braaten for being my second reader. I’d also like to thank the Colgate University Neuroscience
Program for the support and funding of this study. A special thank you to my research partner,
Sam Frank, as well as fellow research assistants Alec Hyde, Hillary Mulder, Lillie Laiks, and
Lauren Kasparson for their help with data collection. And lastly, this would not have been
possible without the hard work of Cindy Baker and Adine Schoonmaker in the animal quarters
who kept our rats healthy throughout the experiment.

NEOCLOMIPRAMINE RAT MODEL VALIDATION
Abstract
Obsessive-compulsive disorder (OCD) is a debilitating disease that warrants investigation
because there is currently no effective treatment due to the ambiguous physiological
underpinnings of the condition. Previous research has demonstrated the importance of serotonin
in this disorder, due to the efficacy of treatment with selective serotonin reuptake inhibitors
(SSRI), as well as effects in animal models induced by drugs that target the serotonin system. We
chose to further investigate the clomipramine-induced rat model. Clomipramine is a drug that
prevents the reuptake of serotonin and has been demonstrated in the past to cause OCD-like
symptoms in rats when they are exposed during a sensitive period starting at postnatal day 9 and
continuing through postnatal day 16. We tested the effects of neonatal exposure to clomipramine
on the behavior of rats in a marble open field test and in a hole board, following administration
of either the serotonin agonist, mCPP, or the serotonin antagonist, mianserin. We found a
significant main effect of neonatal exposure for the number of marbles buried, but not for other
behaviors examined. We did find a significant interaction effect for the number of marbles
checked, and found that there was a significant difference between the numbers of marbles
checked between rat types after exposure to mianserin. This study added to the face validity of
the neoclomipramine model, and added to the predictive validity of the behavioral assays used.
Future researchers should ensure their sample sizes are sufficiently large to tease out significant
main effects of neonatal exposure, and should attempt to further elucidate the neurophysiological
basis of this disorder by testing newer, more selective drugs on this model.
Keywords: obsessive-compulsive disorder, clomipramine, mCPP, mianserin, hole poke,
marble burying
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Evaluation of the Validity of a Neoclomipramine Rat Model for Obsessive-Compulsive Disorder
Using the Hole Poke Board and Marble Arena Behavioral Assays
Obsessive-compulsive disorder (OCD) is diagnosed when someone has obsessions,
consisting of repulsive thoughts or images, as well as compulsions, defined as repeated behaviors
or mental acts that a person is compelled to do, often in an attempt to satiate the obsession. It has
been suggested that compulsions arise from a deficient response feedback from an action that an
individual carries out to address the obsession (See review by Joel & Avisar, 2001). This creates
a problem because the completion of the compulsive behavior does not result in the cessation of
the obsession due to the person’s dissatisfaction resulting from the deficient or complete lack of
feedback. According to the most recently published Diagnostic and Statistical Manual of Mental
Disorders, symptoms must cause significant distress that leads to social, occupational, or other
impairments in daily functioning in order for someone to be diagnosed as having OCD (5th
ed.;
DSM-V; American Psychiatric Association, 2013).
Because OCD is often comorbid with other anxiety, mood, impulse-control, and
substance abuse disorders, the likelihood of clinicians diagnosing false positive or false negative
cases of OCD is high (Spitzer & Wakefield, 1999). A false positive occurs when someone is
wrongly classified as having a disorder through the incorrect application of the DSM criteria, and
a false negative occurs when people with a true disorder are diagnosed as having no problem.
Data from the National Comorbidity Survey Replication results from the early 2000s, suggests
that lifetime prevalence for DSM-IV OCD is 2.3%. This is low considering 28.2% of
respondents that were examined experienced obsessions or compulsions at some point in their
life (Ruscio, Stein, Chiu, & Kessler, 2010). Checking, hoarding, and ordering are the three most
common obsessions /compulsions, but most respondents experienced just one out of the nine
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obsession/ compulsion types examined (Ruscio et al., 2010). Sociodemographic predictors for
the disorder include age, where 18-29 year olds are most commonly diagnosed, and sex, where
females are more commonly diagnosed as adults but males are more commonly diagnosed as
children (Ruscio et al., 2010). It is difficult to determine the long-term persistence rate of
pediatric diagnoses of OCD, but estimates have found a rate of 41% for diagnoses of full OCD
and 60% for diagnoses of full or subthreshold OCD (Stewart et al., 2004). A more recent review
stated that those who are diagnosed typically spend about nine years living with OCD, an
estimate for persistence that is lower than originally thought (Ruscio et al., 2010). Even though
the persistence rate is not terribly long, the effectiveness of current available pharmacological
treatments is low, which makes the investigation of OCD a necessity.
OCD warrants investigation for many reasons, the biggest of which is that the
neurobiological basis is still largely unknown, making it difficult to develop targeted treatment
strategies. The most effective treatments found so far have been response exposure and
prevention, but these still only help 70% of people with OCD (See review by Albelda & Joel,
2012). Early research on pharmacological treatments found responses of people with OCD to
serotonergic and norepinephrinergic uptake inhibitors such as clomipramine (Ruscio et al.,
2010). Treatment then moved towards using selective serotonin reuptake inhibitors (SSRIs), such
as citalopram, fluoxetine, fluvoxamine, sertraline, and paroxetine, due to their greater
tolerability, though they are not any more effective (Bergeron et al., 2002). Further support for
the role of serotonin in causing OCD symptoms comes from studies that have shown mCPP, a
serotonin 1A, 1D, and 2C receptor agonist, exacerbates OCD in humans, and mianserin, a
serotonin 5-HT2c receptor antagonist, alleviates symptoms (See review “Obsessive-Compulsive,”
2014). Mirtazapine, a serotonergic antagonist, has been found to antagonize the effects of mCPP,
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further implicating the role of serotonin in causing OCD symptoms (Mckie et al., 2011).
Treatment studies have also demonstrated the ineffectiveness of non-SRI antidepressants (such
as desipramine), anxiolytics (such as diazepam), anti-epileptics, and adrenergic agonists,
distinguishing OCD from other anxiety and tic disorders (See review “Obsessive-Compulsive,”
2014; “Medications,” 2014). Based on these current and previously attempted pharmacological
treatments, the neurotransmitter system that has been implicated as playing the biggest role in
causing OCD symptoms is serotonin.
There are fourteen subtypes of serotonin (5-HT) receptors, and it has been difficult for
researchers to identify which ones play a role in causing specific mental states. The knowledge
that mCPP is a 5-HT2c receptor agonist though implicates the serotonin 2C receptor in mediating
the factors that contribute to an OCD brain state. The 5-HT2c receptor is thought to be
downregulated by SSRI treatment, leading to disinhibition of the mesolimbic dopamine system
and a generalized anti-anxiety effect on the brain (Serretti, Artioli, & De Ronchi, 2004). Specific
brain regions found, in one study, to have increased Fos-like immunoreactivity induced by
systemic administration of mCPP included the central nucleus of the amygdala, the bed nucleus
of the stria terminalis, the lateral septum, paraventricular nucleus of the hypothalamus, lateral
hypothalamus, infralimbic, and prelimbic cortex in rats (Singewald, Salchner, & Sharp, 2003).
These correspond with brain regions that have been implicated in imaging studies of OCD
patients (See review Albelda & Joel, 2012). These regions are spread out over the whole
anatomy of the brain and have numerous roles in carrying out various functions, but this list is
helpful in determining the possible efficacy of animal models, which can provide a means to
study the biological bases of diseases in instances where human models would be unethical.
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Although no one model can perfectly mimic the symptoms of the disorder it is
representing, creating models with viable validity can help elucidate the unknown aspects of the
neurobiological basis of a disorder such as OCD. Researchers and clinicians have categorized
humans with OCD into four behavioral groups: aggressive sexual and religious obsessions with
checking compulsions; symmetry obsessions with compulsions of classification, sorting, and
repetitiveness; obsessions with contamination and subsequent cleaning compulsions; and
hoarding (Boulougouris, Chamberlain, & Robbins, 2009). Due to the wide variety of possible
combinations of obsessions and compulsions, no one animal is going to be able to perfectly
model the human condition: models usually emulate one or a maximum of two of these different
classifications of OCD symptoms.
In an ideal situation, an animal model should demonstrate face validity, predictive
validity, construct validity, and reliability (See review Albelda & Joel, 2012). Face validity is
whether the model accurately represents a human physiological state, and is often seen in
behavioral tests. In the case of OCD, an example of expected animal behavior is repetitive or
compulsive checking. Predictive validity is whether we can predict, based on the results of
experiments with the model, if treatment with a specific drug will be effective in humans. For
example, in the case of OCD, this would be seen in the model’s positive response to SSRIs but
negative response to other classes of drugs such as anxiolytics and non-serotonergic
antidepressants that are ineffective in humans with OCD. Construct validity is a similarity in
etiology of the disorder. Face, as well as construct, validity is less important because many
models are made for the purpose of discovering the physiological basis of a disorder. Reliability
and predictive validity, on the other hand, are both crucial to establishing a solid model
(Boulougouris, Chamberlain, & Robbins, 2009; Albelda & Joel, 2012). Past OCD model types
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that have shown potential include genetic, pharmacologically induced, and behavioral models.
Behavioral and genetic models that relate to serotonin include the 5-HT7 and 5-HT2c knockout
mouse models, respectively.
One genetic model, the 5-HT7 knockout mouse, was developed in an attempt to
demonstrate a treatment for OCD behavior as exhibited by the marble burying assay, which has
been used as a behavioral model for OCD in mice. Knockout mice buried less marbles compared
to their wild-type siblings (Hedlund & Sutcliffe, 2007). Success of the knockout mouse model
was confirmed when acute administration of a 5-HT7 receptor antagonist in a wild-type mouse
produced similar marble burying results as for the knockout mice; wild type mice treated with
the 5-HT7 receptor antagonist buried significantly fewer marbles than wild type mice. These
results suggest possible predictive validity for a treatment model, but do not demonstrate face
validity because they are using a baseline behavior as a measure of OCD and looking for a
reduction, rather than an exacerbation, of marble burying as compared to controls.
Another genetic serotonin receptor model is the 5-HT2c knockout mouse model. This
model demonstrated face validity through the discovery that knockout mice had greater non-
ingestive chewing of clay compared to controls, showed a distinct, more ordered screen chewing
pattern compared to controls, and were slower to habituate to a head-dip board than controls
(Chou-Green et al., 2003). There were no overall behavioral deficits seen in the knockout mice,
suggesting that the differences between knockout mice and controls were not secondary to
sensory and motor disruption (Chou-Green et al., 2003). Though the face validity of this model
fits with that of other OCD models, the 5-HT2c knockout mouse model does not demonstrate
appropriate construct validity because pharmacologically-induced models suggest that OCD
results in part from hypersensitive 5-HT2c receptors, rather than inactive ones. Other genetic
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models have targeted the enhancement of the dopamine D1 receptor and the deletion of the
Sapap3 excitatory postsynaptic scaffolding proteins, but the construct validity for these models is
even less clear than for these two serotonin receptor knockout mouse models. In addition to these
genetic models, pharmacologically induced models also support the role of serotonin in causing
an OCD brain state.
mCPP is a 5-HT receptor agonist that has been found to lead to compulsive checking and
ritual-like motor behaviors in rats. Chronic treatment with fluoxetine, but not acute treatment of
diazepam or chronic treatment of desipramine, blocked mCPP-induced directional persistence in
a reinforced spatial alternation task (Boulougouris, Chamberlain, & Robbins, 2009). In another
model pharmacologically induced by mCPP, acute treatment with mianserin, as well as chronic
treatment with clomipramine and fluvoxamine, blocked ritualistic chewing behavior, though
treatment with haloperidol, a dopamine antagonist, and diazepam, a GABA agonist, did not
(Kreiss et al., 2013). A recent study that examined the effects of 5-HT2a and 5-HT2c receptor
antagonism on mCPP-induced spontaneous alternation in male Wistar rats found that a 5-HT2c
but not a 5-HT2a receptor antagonist significantly reduced mCPP-induced directional persistence
(Papakosta et al., 2013). This model demonstrates possible predictive validity due to the similar
role of the 5-HT2c receptor in causing the OCD-like behavior in rats as well as OCD behavior in
humans. Another study, conducted by Cornelio and Nunes-de-Souza (2006), found increased
behavioral indices of anxiety in mice with mCPP injected into their amygdaloid complex, and
this increase was then blocked by local injection of a 5-HT2c receptor antagonist. Although these
models have demonstrated predictive validity and possible construct validity, they have not
shown strong reliability or face validity.
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Another way to pharmacologically induce OCD in an animal model is with neonatal
exposure to clomipramine. We chose to use the clomipramine-induced model of OCD because it
has shown in the past to have face as well as predictive and possible construct validity. We were
interested in further elucidating this phenomenon – why a drug used to treat OCD in some cases
in humans can cause permanent neurobiological changes in brain state resulting in OCD-like
tendencies when injected into the brains of neonatal rats. The model was first proposed by
Andersen, Greene-Colozzi, and Sonntag in 2010. They first performed intraperitoneal injections
of either saline or 15 mg/kg clomipramine in the rats. This was followed seven hours later by a
second injection, for a total of sixteen injections across an eight-day period on postnatal days 9-
16. The researchers then tested the rats on six behavioral assays starting on postnatal day eighty-
five. The tests included the elevated plus maze (EPM), marble burying, spontaneous alternation
in a t-maze, reversal learning in a four-arm maze, the win-shift paradigm, and caching (hoarding)
behavior. They found that the clomipramine rats spent significantly more time in the closed arm
of the EPM, buried more marbles than vehicles when placed into an open field, required more
trials to reach criterion in reversal learning, required more days to initially learn task in the win-
shift paradigm, and cached significantly more food pellets. These results demonstrate the face
validity of this model for compulsive as well as anxious behaviors. The researchers also found
increased mRNA expression for 5-HT2c receptors in the orbital frontal cortex, demonstrating the
possible construct validity of this model. These behavioral and physiological similarities to
humans with OCD contribute to the efficacy of these clomipramine rats as a model for multiple
OCD-like behaviors.
Previous Colgate studies have also used the clomipramine model, and coined the terms
neoclomipramine and neosaline to distinguish between the rat types. In the spring semester of
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2013, senior research students found that neoclomipramine rats buried and checked significantly
more marbles, entered significantly fewer total arms and open arms, spent significantly more
time in closed arms in an elevated plus maze, and had a significantly higher poke/hole ratio than
neosaline controls (Bavley, 2013; Belluardo, 2013, & Rettstatt, 2013). These results again
demonstrate the face validity of the neoclomipramine model for compulsive (marble burying,
hole pokes) as well as anxious (elevated plus maze) behaviors. Chronic administration of
fluoxetine, an SSRI used to treat OCD in humans, normalized the amount of time
neoclomipramine rats spent in closed arms of the elevated plus maze as well as the poke/hole
ratio of neoclomipramine rats for the hole board (Belluardo, 2013; Rettstatt, 2013). Chronic
administration of desipramine, a drug that can supplement SSRI treatment for humans with
OCD, but is not an effective treatment when given alone, had no effect on neoclomipramine rats
for any behavior tested (Rettstatt, 2013). These results support the multiple validities of the
neoclomipramine rat model for OCD because they demonstrate the rats having a permanently
altered brain state that results in behavior resmebling humans with OCD, not just anxious
behavior.
Our chosen behavioral assays include the hole board and the open field test with marbles.
These are both behavioral tests that are well known within the field of anxiety disorder research.
The operational definition of compulsiveness in a hole poke board test is a high calculated
poke/hole ratio because this is a reflection of repetitive behavior, similar to how OCD manifests
itself in many humans. We recorded the total number of pokes, the location of each head poke,
and the order in which the head pokes happened. This behavioral assay has been shown to be a
valid measure of rats’ levels of compulsiveness and exploration, and can demonstrate the balance
between a curiosity-based approach to a stimulus and a fear-based avoidance of a novel stimulus.
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It is often difficult to distinguish between these two phenomena though because many behaviors,
especially those in response to a new environment, will bring out both neophilia and neophobia
(Brown & Nemes, 2008). Open field tests with marbles have also been shown to be a useful
measure of OCD-like symptoms, in which rats appear to be less affected by the novelty-of-
environment phenomenon (Thomas, Burant, Bui, Graham, Yuva-Paylor, & Paylor, 2009). In our
study, we looked at checking behavior, which involves the rat simultaneously sniffing and
pawing a marble, as well as total number of buried marbles. Previous studies have found no
significant differences in the number of marbles buried by mice exposed to the marbles for one
day prior to testing, five days prior to testing, or with no exposure prior to testing, suggesting this
is a robust measure of repetitive digging behavior rather than a measure of novelty-induced
anxiety (Thomas et al., 2009; Review by Albelda & Joel, 2012). Marble burying has been found
to decrease after an animal is exposed to chronic administration of SSRIs at doses that do not
affect locomotor activity, which points to this being an assay with some predictive validity, but
findings from studies looking at the effects of anxiolytics are mixed, which detracts from the
overall predictive validity (See review by Albelda & Joel, 2012). The methods for this marble
burying behavioral assay were taken from Andersen, Greene-Colozzi, and Sonntag (2010), who
found that clomipramine rats buried significantly more marbles than saline controls when placed
into the open field test. Because we were attempting to further validate the clomipramine-
induced OCD model, we decided the open field marble-burying test and hole board were better
choices for bringing out compulsive behavior, but we did not examine the elevated plus maze
behavior because it is a test for generalized anxiety, rather than OCD.
We chose to use mCPP and mianserin in our testing phases because both of those drugs
have known effects on humans with OCD. Clinical studies have found that mCPP, a serotonin
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agonist, can produce anxiety and can exacerbate symptoms in humans that already have panic or
OCD disorders (Dickstein, Riba, & Oldham, 1996; Shanks, 2001). In contrast, mianserin, a
serotonergic antagonist used in humans as a supplement treatment, has been found to alleviate
OCD symptoms (Hood et al., 2008).
In the present investigation, we sought to validate a clomipramine-induced OCD rat
model by testing drugs that have known effects in humans with the disorder, on twenty-nine rats
(fifteen neo-salines and fourteen neo-clomipramines). We had four drug conditions, including a
saline control, a 0.4 mg/kg mCPP dose, a 0.8 mg/kg mCPP dose, and a 3mg/kg mianserin dose.
We evaluated the behavior of the rats using a 4x4 hole-poke board and an open field marble test.
We measured total pokes, total holes poked, and pokes/hole made in a five minute time period,
and total marble checks, percentage checks made in the first half of the trial, total marbles
buried, and number of marbles carried in a fifteen minute time period.
Methods
Subjects (Animals)
Twenty-nine male Sprague-Dawley rats were born in five litters on July 26 or on July 28,
2013 at Colgate University in Hamilton, NY. All procedures were approved by the Colgate
University Animal Care and Use Committee Protocol 13-8R and were consistent with the
National Research Council (1996, 2003). The animals were weaned on postnatal day twenty-
four, and were housed in cages, in groups of two litter-mates, under a twelve hour light/dark
cycle. The animals had free access to food and water during the entirety of the experiment.
Animals were handled and weighed biweekly during scheduled cage changes to minimize stress
during the testing periods.
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Drug exposure
Neonatal exposure
Beginning on postnatal day nine and continuing through postnatal day sixteen, rats were
given intraperitoneal injections of either saline (0.9% NaCl, n = 15) or clomipramine (15 mg/kg,
n = 14). Clomipramine was prepared with saline (0.9% NaCl) such that the proper dosage was
administered as 1 mg/mL. These injections were repeated twice daily, seven hours apart, for a
total of sixteen injections across eight days.
Adult drug administration
Beginning on postnatal day eighty, October 16th
, and continuing through postnatal day
one hundred-six, November 9th
, all of the adult rats went through three consecutive subcutaneous
injections of saline, followed by four subcutaneous injections of either saline (0.9% NaCl),
mianserin (3 mg/kg), or one of two possible doses of mCPP – a low dose (0.4 mg/kg) and a high
dose (0.8 mg/kg). For the first injection, all of the rats were given acute injections of saline
(0.9% NaCl) and then put back in their home cage so as to allow the rats to get used to being
handled and being injected. For the second and third saline trials, all of the rats were again given
saline and then their behavior in the hole board and marble arena was observed. The same
sequence occurred on the fourth – seventh trials except the rats received an injection of one out
of four possible drugs. The drugs were pseudo-randomly assigned to the rats across the
neoclomipramine and neosaline groups such that each rat received every drug or dosage one time
during those four trials. Both the injector and the behavioral observer were blind to the identity
of the injection. Drugs were made fresh daily and the drug powder was dissolved in saline such
that 1 mL solution/kg body weight was delivered to the animal.
Behavioral testing
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Beginning on postnatal day eighty and continuing through postnatal day one hundred-six,
all of the adult rats underwent behavioral assessment twenty minutes after an acute injection of
either saline or the pseudo-randomly assigned drug. The rat was first placed in the hold board for
five minutes, and then immediately placed in the marble arena adjacent to the hole board for
fifteen minutes such that each rat underwent a total of twenty minutes of behavioral testing. The
rats were then returned to their home cage in the colony room. All behavioral trials were
observed by one research assistant present in the room, and were also digitally recorded.
Behavioral trial days assessed the effects of a single administration of one of the drugs, and were
conducted forty-eight hours apart for a total of two saline and four drug trial testing days for each
group of eight rats.
Apparatus
Hole Board
The hold board was a 60 cm by 60 cm square open box, and contained sixteen evenly
spaced holes on the floor of the apparatus. Each hole had a diameter of approximately four cm,
allowing the rats to poke their heads through but not their entire bodies. The box had corner
supports that put its height at about 6 cm above the ground, allowing the rats to have space to
fully poke their heads through.
A poke was counted when the rat’s head broke the bottom plane of the board. The
assessment included recording the total number of holes poked in the first versus second half of
the five-minute trial, as well as the location of each poke on the board.
The apparatus was wiped down with 70% isopropyl alcohol between the assessments of
each rat, so as to remove any confounding odors from the previous rat.
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Marble Arena
The marble arena was a plastic bin measuring twenty-nine inches long by seventeen and a
half inches wide by fifteen and a half inches deep and filled with four to five centimeters of
bedding. Twenty-four marbles were evenly spaced in a four by six pattern.
The number of marble checking behaviors and the numbers of marbles buried were
counted for each rat. Buried marbles were recorded as either “not buried,” “partially buried,” or
“fully buried.” A marble was considered fully buried only if more than 75% of it was under
bedding, and partially buried if between 50% and 75% of the marble was under bedding. Marble
checking behavior occurred when a rat stopped and sniffed a marble while simultaneously
exploring it with its paw. The marble checking behavior was compared between the first half of
the trial and the second half by dividing the segment for each rat into seven and a half minute
observation periods.
The marbles were cleaned with 70% isopropyl alcohol after each pair of cage mates
completed their trials. The same bedding was used only for pairs of rats that also shared a home
cage. The marbles were replaced in the original formation after each trial.
Statistical analysis
All data were expressed as mean ± the standard error of the mean (SEM). Statistical
analyses of the effect of rat type and drug administration were performed using mixed-design
two-way ANOVAs. Statistical analysis of the differences between averages for the saline two
trials and the randomized saline trials were performed using student’s t-tests to ensure there were
not significant differences. All tests were evaluated at p<0.05. P values between 0.05 and 0.08
were considered to be approaching significance and were analyzed using the Tukey HSD test and
still reported.
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Results
Effects of Neoclomipramine Exposure on Behaviors in the Hole Poke Board
Neoclomipramine exposure did not affect total hole pokes
An independent samples t-test that compared the mean number of total hole pokes for the
neoclomipramine rats (M = 21.00, SEM = 2.01) and neosaline rats (M = 18.87, SEM = 2.18)
during saline trial two was not found to be statistically significant.
The number of hole pokes data was subjected to a mixed-design two-way analysis of
variance (ANOVA) having four levels of drug administration (randomized saline, low dose
mCPP, high dose mCPP, and mianserin) and two levels of rat type (neoclomipramine and
neosaline). No effect was found to be statistically significant, but the main effect of drug
administration approached significance, yielding an F ratio of F(3, 81) = 2.58, p = 0.06. A Tukey
HSD test revealed that the mean total hole pokes for rats treated with mianserin was significantly
lower than the mean total hole pokes for rats treated with randomized saline. The mean for rats
treated with low dose mCPP did not differ significantly from the means of rats treated with
randomized saline, high dose mCPP, or mianserin, and the mean for rats treated with high dose
mCPP did not differ significantly from the means of rats treated with randomized saline or
mianserin.
Percentage Pokes in the First Half
An independent samples t-test that compared the mean percentage of pokes in the first
half of the trial for the neoclomipramine rats (M = 0.60, SEM = 0.03) and the neosaline rats (M =
0.58, SEM = 0.03) during saline trial two was not found to be statistically significant.
Percentage of pokes in the first half data was subjected to a mixed-design two-way
ANOVA having four levels of drug administration (randomized saline, low dose mCPP, high
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dose mCPP, and mianserin) and two levels of rat type (neoclomipramine and neosaline). Only
the main effect of drug administration was found to be statistically significant yielding an F ratio
of F(3, 81) = 4.88, p < 0.01. A Tukey HSD test revealed that the mean for rats treated with low
dose mCPP (M = 0.59, SEM = 0.03) was significantly lower than the mean for rats treated with
mianserin (M = 0.72, 0.03), p < 0.01. There was a trending difference between the means for rats
treated with high dose mCPP (M = 0.73, SEM = 0.03) and rats treated with low dose mCPP, p =
0.057. The average percentage of pokes in the first half scores for rats treated with either low
dose mCPP, high dose mCPP, or mianserin did not significantly differ from scores of rats treated
with randomized saline, and high dose mCPP scores did not significantly differ from mianserin
scores (Figure 1).
Figure 1. Mean percentage of hole pokes collapsed across neosaline and neoclomipramine rats
during testing treatment with saline, low and high doses of mCPP, and mianserin. The pound
sign indicates a trending significant difference between the average percentage hole pokes of rats
treated with high dose mCPP and rats treated with low dose mCPP. The asterisks indicate a
significant difference at the 0.01 level between the average percentage of hole pokes for rats
treated with mianserin and rats treated with low dose mCPP.
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Total Holes Used
An independent samples t test that compared the mean number of total holes used by
neoclomipramine rats (M = 11.14, SEM = 0.71) and neosaline rats (M = 10.93, SEM = 0.87) was
found to not be statistically significant.
The number of total holes used data was subjected to a mixed-design two-way ANOVA
having four levels of drug administration (randomized saline, low dose mCPP, high dose mCPP,
and mianserin) and two levels of rat type (neoclomipramine and neosaline). No effect was found
to be statistically significant, but the main effect of drug administration approached significance,
yielding an F ratio of F(3, 81) = 2.46, p = 0.069. A Tukey HSD test revealed that there were no
significant differences between the means of rats treated with any drug.
Pokes/Hole
An independent samples t-test that compared the mean number of pokes/hole for the
neoclomipramine rats (M = 1.85, SEM = 0.09) and neosaline rats (M = 1.66, SEM = 0.11) during
saline trial two was not found to be statistically significant.
The number of pokes/hole data was subjected to a mixed-design two-way analysis of
variance having four levels of drug administration (randomized saline, low dose mCPP, high
the main effect of drug was found to be statistically significant.
The data failed Mauchly’s test of sphericity so results included were based on the Huynh-
Feldt test. The main effect of drug administration yielded an F ratio of F(2.90, 78.31) = 3.61, p <
0.05. A Tukey HSD test revealed that the mean number of pokes/hole for rats treated with low
dose mCPP (M = 1.90, SEM = 0.06) was significantly higher than the mean number of
pokes/hole for rats treated with mianserin (M = 1.62, SEM = 0.07), p < 0.01. The means for rats
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treated with low dose mCPP, high dose mCPP, and mianserin did not differ significantly from
the mean for rats treated with randomized saline. The mean for rats treated with low dose mCPP
did not differ significantly from the mean for rats treated with high dose mCPP, which did not
differ significantly from the mean of rats treated with mianserin (Figure 2).
Figure 2. Mean number of pokes/hole collapsed across neosaline and neoclomipramine rats
during testing treatment with saline, mCPP, and mianserin. The asterisks indicate a significant
difference at the 0.01 level between the average pokes/hole for rats treated with 0.4 mg/kg mCPP
and rats treated with mianserin.
Repeats
An independent samples t-test that compared the mean number of repeats for the
neoclomipramine rats (M = 9.86, SEM = 1.46) and neosaline rats (M = 7.93, SEM = 1.45) during
saline trial two was found to not be statistically significant.
The number of repeats data was subjected to a mixed-design two-way analysis of
dose mCPP, and mianserin) and two levels of rat type (neoclomipramine and neosaline). No
effects were found to be statistically significant, but the main effect for drug administration
approached significance, yielding an F ratio of F(3, 81) = 2.68, p = 0.053. A Tukey HSD test
revealed that the mean for rats treated with low dose mCPP (M = 9.45, SEM = 0.90) was
19

significantly higher than the mean for rats treated with mianserin (M = 6.37, SEM = 1.03), p <
0.05. The difference between the means for rats treated with randomized saline and rats treated
with mianserin approached significance, p = 0.062. There were no significant differences
between the means for rats treated with randomized saline and low or high dose mCPP, the
means for rats treated with low dose versus high dose mCPP, or the means for rats treated with
high dose mCPP and mianserin.
Effects of Neoclomipramine Exposure on Behaviors in the Open Field Test with Marbles
Total Marbles Buried
An independent samples t-test that compared the mean number of marbles buried for the
neoclomipramine rats (M=4.32, SEM =0.82) and neosaline rats (M = 2.23, SEM = 0.53) during
saline trial two was found to be statistically significant, t(27) = -2.09, p < 0.05, indicating that
neoclomipramine rats buried significantly more marbles than neosaline rats (Figure 3).
Neoclomipramine rats buried approximately 194% more marbles than neosaline rats.
Figure 3. Mean number of marbles buried during saline trial 2 for neoclomipramine and
neosaline rats. The asterisk indicates a significant difference at the 0.05 level between the
average number of marbles buried by each rat type.
20

The total marbles buried data was subjected to a mixed-design two-way analysis of
the main effect of rat type was found to be statistically significant.
The main effect of rat type was such that the number of marbles buried by
neoclomipramine rats (M = 4.24, SEM = 0.42) was significantly greater than the number of
marbles buried by neosaline rats (M = 2.36, SEM = 0.41), F(1, 27) = 10.32, p < 0.01 (Figure 4).
Neoclomipramine rats buried 180% more marbles than neosaline rats.
Figure 4. Mean number of marbles buried by neoclomipramine and neosaline rats during testing
with saline, low and high doses of mCPP, and mianserin. The asterisk indicates the main effect
of rat type in that neoclomipramine rats (left bars) buried significantly more marbles than
neosaline rats (right bars).
Total Marbles Checked
An independent samples t-test that compared the number of marbles checked for
during saline trial two was not found to be statistically significant.
21

The total marble checks data was subjected to a mixed-design two-way analysis of
dose mCPP, and mianserin) and two levels of rat type (neoclomipramine and neosaline). The
main effect of drug and the interaction effect were found to be statistically significant.
The main effect of drug yielded an F ratio of F(3, 81) = 24.02, p < 0.001. A Tukey HSD
test revealed that the mean for rats treated with randomized saline (M = 17.25, SEM = 2.76) was
significantly higher than the means for rats treated with low dose mCPP (M = 3.49, SEM = 0.68),
p < 0.001, and high dose mCPP (M = 1.36, SEM = 0.27), p < 0.001, and was trending towards
being significantly higher than rats treated with mianserin (M = 9.89, SEM = 1.36), p = 0.058.
The means for rats treated with low dose mCPP and mianserin were significantly higher than the
mean for rats treated with high dose mCPP, p < 0.05 and 0.001 respectively. The mean for rats
treated with mianserin was significantly higher than the mean for rats treated with low dose
mCPP, p < 0.01 (Figure 5).
Figure 5. Mean number of marbles checked collapsed across Neoclomipramine and neosaline
rats during treatment with saline, low and high doses of mCPP, and mianserin. The asterisks over
the first bar indicate significant differences between rats treated with saline and rats treated with
both low dose and high dose mCPP at the 0.001 level. The pound sign indicates a trending
22

difference between rats treated with saline and rats treated with mianserin. The asterisk over the
second bar indicates a significant difference between rats treated with low dose mCPP and high
dose mCPP at the 0.05 level. The asterisks over the fourth bar indicate significant differences
between rats treated with mianserin and rats treated with low dose mCPP at the 0.01 level and
between rats treated with mianserin and rats treated with high dose mCPP at the 0.001 level.
The interaction effect, F(3, 81) = 3.57, p < 0.05, was analyzed using four separate
independent samples t-tests comparing rat type to each possible level of drug administration. An
independent samples t-test that analyzed the difference between number of marbles checked for
when treated with randomized saline was not found to be statistically significant. An independent
samples t-test that analyzed the difference between number of marbles checked for
neoclomipramine rats (M = 2.79, SEM = 0.79) and neosaline rats (M = 4.20, SEM = 1.07) when
treated with low dose mCPP was not found to be statistically significant. An independent
treated with high dose mCPP was not found to be statistically significant. An independent
treated with mianserin was found to be approaching significance, t(27) = -2.02, p = 0.053 (Figure
6).
23
Drug Condition
Randomized Saline Low dose mCPP High dose mCPP Mianserin

Figure 6. Mean number of marbles checked by Neoclomipramine and neosaline rats when
treated with saline, low dose mCPP, high dose mCPP, or mianserin. Pound sign indicates
trending significant difference between the average number of marbles checked by
Neoclomipramine and neosaline rats when treated with mianserin. Demonstrates interaction
effect.
Percentage of Marbles Checked in the First Half
An independent samples t-test that compared the percentage of marbles checked in the
first half of the trial for neoclomipramine rats (M = 0.54, SEM = 0.06) and neosaline rats (M =
0.63, SEM = 0.06) was not found to be statistically significant.
The percentage of marbles checked in the first half data was subjected to a mixed-design
two-way analysis of variance having four levels of drug administration (randomized saline, low
dose mCPP, high dose mCPP, and mianserin) and two levels of rat type (neoclomipramine and
neosaline). No effects were found to be statistically significant.
Discussion
To review, this study further evaluated the face and predictive validity of a new model for
obsessive-compulsive disorder consisting of neonatally exposing rats to clomipramine.
Behaviors chosen for evaluation of the face validity include marble burying and checking as well
as hole poke behaviors. The serotonergic agonist mCPP and serotonergic antagonist mianserin
were selected for the evaluation of the predictive validity of the model, since the effects of these
drugs on human patients with OCD are known. mCPP has been observed as worsening the
symptoms of humans with OCD, while adding general anxiety to some people without OCD,
whereas mianserin has been observed as alleviating the symptoms of humans with OCD.
Marble Open Field Test Behaviors
Marble Burying
24

For the marble burying, if the model had exhibited face validity, we would have expected
to see neoclomipramines burying more marbles than neosalines at baseline. If the model had
exhibited predictive validity, we would have expected to see differences between the neonatal
groups following administration of mCPP. Neoclomipramines would bury more marbles,
whereas neosalines would be unaffected. This is because mCPP has been shown to exacerbate
the symptoms of individuals with OCD, but not to increase obsessions or compulsions in healthy
controls. On the other hand, after administration of mianserin, we would have expected to see
neoclomipramines bury less marbles and the neosalines once again unaffected, and thus see less
of a difference between number of marbles buried for the two rat types as compared to baseline
or mCPP.
Our results demonstrated a main effect of neonatal exposure, but not a main effect of
drug exposure or an interaction effect between the two. This does in fact fit with previous
findings that marble burying has good face validity, but poor predictive validity (See review
Albelda & Joel, 2012). The main effect for rat type demonstrates face validity of the model in
that the behavior between neoclomipramines and neosalines was significantly different, but the
absence of a main effect of drug or interaction effect demonstrates poor predictive validity
because the marble burying behavior did not respond to either dose of mCPP or mianserin,
unlike the other behavioral tests examined.
A study carried out by senior research students at Colgate University in spring 2013
under the guidance of Dr. Deb Kreiss found that neoclomipramine rats buried significantly more
marbles than neosaline rats prior to drug exposure (Bavley, 2013). These results suggest that the
behavioral assay has excellent face validity. Our results also replicate the findings of Andersen,
25

Greene-Colozzi, and Sonntag, (2010) who were the first to find that neoclomipramine rats bury
significantly more marbles than controls.
Marble Checking
For marble checking behavior, face validity would have shown neoclomipramines
performing significantly more checks than neosalines at baseline. Predictive validity would have
shown neoclomipramines performing a greater number of checks than their baseline and thus a
bigger difference between the number of checks for neoclomipramines and neosalines after
administration of mCPP. It also would have shown neoclomipramines performing fewer checks
than their baseline and thus a smaller difference between the number of checks for
neoclomipramines and neosalines after administration of mianserin.
Our results demonstrated a main effect of drug and an interaction effect with a significant
difference between rat types following administration of mianserin, when neoclomipramine rats
performed a greater number of checks than neosaline rats. These results do not support the model
having face validity, as is demonstrated by the results having a lack of a significant main effect
of rat type, as well as by exhibiting the opposite result of what we were expecting following the
administration of mianserin. In comparison with a previous study, we did not find that
neoclomipramine rats checked significantly more marbles than neosaline rats prior to drug
exposure (Bavley, 2013). There is some predictive validity though as there was a main effect for
drug type as well as an interaction effect. Across both rat types, the mean for mianserin was
significantly higher than the mean for low as well as high dose mCPP; the mean for low dose
mCPP was significantly higher than the mean for high dose mCPP; and the mean for baseline
saline was significantly higher than the means for all other drug conditions. This is a highly
variable behavior that is therefore greatly affected by a small sample size. This very possibly
26

influenced the results of the present study as compared to the previous spring 2013 study that
used two times as many rats.
Percentage Checks in the First Half
Novelty-induced anxious behavior as evaluated by the percentage marbles checked in the
first versus second half of the trial is not well studied, so we did not have strong expectations of
any particular changes occurring. For the model to have exhibited face validity, we would have
expected neosalines to have a higher percentage of checks in the first half of the trial than in the
second half, and we would have expected neoclomipramines to exhibit one of two possible
behaviors: either to have no differences between percentage of checks in the first versus second
half of the trial, or to have performed a higher percentage of checks in the second half of the
trial. This is based on the notion that novelty-induced anxiety in rats exhibits itself immediately
(which can be operationally defined as in the first half of trials) whereas OCD-like compulsive
/repetitive behavior is consistent throughout both halves of the trial (which can be operationally
defined as having equal percentage checked in both halves of the trial or a higher percentage
checked in the second half of the trial as compared to the first).
For the model to have exhibited predictive validity, we would have expected mCPP to
either have no effect on the neoclomipramine ratio of percentage checks in the first versus
second half, or for mCPP to increase the percentage of marbles checked in the second half. This
is based on mCPP’s known effects of exacerbating OCD symptoms in humans. In contrast, we
would have expected mianserin to reduce the difference, in the second half of the trial, between
the percentages of marbles checked by neoclomipramine and neosaline rats. This is based on
mianserin’s known effects of alleviating OCD symptoms in humans.
27

Our results demonstrated no significant findings, which is not consistent with previous
research. Previous findings demonstrate neoclomipramine rats performed a significantly higher
percentage of checks in the second half of trials whereas neosaline rats performed a significantly
higher percentage of checks in the first half of trials (Bavley, 2013).
Hole Board Test Behaviors
Pokes/Hole
The poke/hole ratio is one of the most important pieces of information that comes out of
the hole board data set for any experiment on OCD using that behavioral assay. For the results to
have shown face validity, we expected to see neoclomipramines having a significantly higher
baseline ratio than neosalines. For the results to have shown predictive validity, we expected to
see mCPP exacerbating neoclomipramine symptoms and thus increasing the difference between
the ratios of the two rat types, but not necessarily affecting the poke/hole ratio of neosaline rats.
We would have expected to see mianserin alleviating the symptoms of neoclomipramine rats and
thus decreasing the difference in poke/hole ratio between the rat types.
We only found a main effect of drug, which does not fit with previous findings. A
previous study found that neoclomipramine rats had a significantly higher poke/hole ratio than
neosaline rats prior to drug exposure (Belluardo, 2013).
Repeats
As with the poke/hole ratio, predictions about this behavior that would exhibit face
validity include that the number of repeats should have been higher for neoclomipramine rats
than for neosaline rats at baseline. Results that would indicate predictive validity for this model
include that mCPP should have caused the difference between neoclomipramine rats and
neosaline rats to increase, due to increasing the number of repeats performed by
28

neoclomipramine rats. We would have expected mianserin to have reduced or eliminated the
significant difference between the number of repeats by neoclomipramine rats and neosaline rats
by decreasing the number of repeats carried out by neoclomipramine rats.
As with the pokes/hole ratio, we only found a main effect of drug, and there is nothing
from previous literature to compare to because no one has examined this particular aspect of the
hole board data in previous studies. Although the analysis was not performed, we would also
expect the repeat data to correlate with the pokes/hole data because a higher pokes/hole ratio
indicates a greater amount of repeats.
Total Pokes Made
For total pokes, predictions for the model demonstrating face validity would be that the
baseline results could demonstrate differences between the two rat types with neoclomipramine
having an overall increase in total pokes, or demonstrate no differences in total checks because
the differences are more apparent in the pokes/hole or repeat data. For the model to demonstrate
predictive validity, we should have seen an increase in total pokes following the administration
of mCPP. After administration of mianserin, we should have seen a decrease in total pokes.
Our results demonstrated only a main effect of drug, which mostly fit with previous
findings that the number of pokes performed by neoclomipramine rats did not differ from that of
neosaline rats (Belluardo, 2013).
Percentage Pokes During the First Half
For percentage of pokes in the first half, predictions that reflect face validity would
include neoclomipramines having an equal or smaller percentage of pokes in the first half of
trials than neosaline rats at baseline. For the model to reflect predictive validity, we would have
looked to see if mCPP exacerbated this effect, and increased the difference between the
29

percentage of pokes in the first half versus second half of the trial (making the percentage in the
second half larger than the percentage in the first half). We also would have looked to see
whether mianserin alleviated the effect and reduced the difference between the percentage of
pokes in each trial half performed by neoclomipramines and neosalines.
We only observed a main effect of drug for this behavioral aspect, and there is nothing in
previous literature to compare to because no one has examined this particular aspect of the hole
board data in previous studies.
Total Holes Used
For total number of holes, there were a couple of different possible predictions for
demonstrating face validity. If the assay measures more of the compulsiveness that is a
characteristic of OCD, then neoclomipramines should have used a significantly higher total
number of holes than neosalines at baseline. If the assay measures the perseverative
characteristic of OCD, then neoclomipramine rats should have used a smaller or equivalent
number of holes as neosalines at baseline because they should return to the same holes more
times than neosaline rats would.
For the model to demonstrate predictive validity, mCPP should not have increased the
number of holes used by neosaline rats, but should have increased number of holes for
neoclomipramine rats if total activity was increased. mCPP should have decreased the number of
holes used if the measurement is based on how many times neoclomipramine rats go back to the
same holes multiple times. Mianserin should not have had an effect on neosaline rats, and it
should have either decreased the number of holes for neoclomipramine rats if the measurement
was based on compulsive activity, or should have increased the number of holes if the
measurement was based on the rats returning to the same hole multiple times.
30

We did not find any significant results, which fit with previous research that found no
significant differences between the total number of holes used by neoclomipramine rats versus
neosaline rats (Belluardo, 2013).
Limitations
Our results did not demonstrate as strong of face and predictive validities as have been
observed in the past with this neoclomipramine model for OCD. There are numerous possible
reasons as to why our results were incongruent with our expectations, and this emphasizes the
arduous and frustrating nature of scientifically validating an animal model.
One factor that could have contributed to individual variation in data for each rat is the
differences among the observers. This covers both the variation within the present study across
the six different research assistants, as well as the variation between the spring 2013 study and
the present study, when there was an entirely new group of research assistants. Other factors
include differences in litter-mates for the rats (some were not housed with siblings due to the
lower number of rats born), different parents of the rats in the spring 2013 study versus the
present study, and the increased number of exposures the rats had to the testing apparatuses in
the present study.
Even though the observers were trained to be as consistent as possible, different people
attend to and focus on different aspects of their environment and this could have lowered the
consistency between the scores of rats being recorded by each of the six possible research
assistants in the lab. Assignments to either the neoclomipramine group or neosaline group were
made randomly to ensure litter-mates had equal numbers in each group, but the housing situation
ended up such that some cage-mates were siblings and some were not. This could have increased
the variability of scores for the different behavioral assays. Increased number of exposures to the
31

testing apparatuses prior to the beginning of the study could have led to less observed behavior,
due to a decrease in the novelty of the environment and thus the exploratory nature of the
animals. Previous research has found that marble burying behavior is resistant to novelty, not
decreasing significantly over the course of five days (Thomas et al., 2009). However, the effect
of novelty on other behaviors we measured is unknown. This would have affected both neonatal
groups the same way though, because all rats had the same number of prior exposures to the
testing apparatuses.
Other factors that limited the results of our study include the use of two hole board
apparatus, one that was handmade and one that was not, which could have contributed to
differences between the present study’s findings and the findings of Kreiss’s research students
from spring 2013. The group in the spring of 2013 only used the professionally constructed hole
board, whereas the present study used both that hole board and one which was handmade by
research assistants in the summer of 2013. The differences between these hole boards could have
contributed to the variability in hole poke behavior in the present study. One factor that could
have influenced the results of the marble burying behavior was the evenness of the bedding in
the marble burying bin. This could have increased the variability of marble burying results
because oftentimes the marbles physically could not be buried in the center, due to a lack of
bedding between the marbles and the floor of the bin. Possible effects of mCPP or mianserin on
additional 5-HT receptor subtypes could have made our results differ from what we expected as
well. There is not much research that distinguishes the effects on individual receptor subtypes of
these various drugs targeting serotonin, but mCPP and mianserin are both less selective than
newer drugs currently available. Looking into the effects of newer, more selective drugs on this
OCD model could be an excellent area for future research. Lastly, our biggest limitation as
32

compared to the sping 2013 study was having a small sample size. This would have increased the
sensitivity of the results to variability and made it less likely for us to have significant findings,
as compared to the spring 2013 study, that had 26 rats per neo-natal group to work with.
Conclusions
We did not observe the face validity of multiple behaviors of the neoclomipramine model
that was documented in a similar study carried out by 2013 senior research students in Kreiss’s
lab at Colgate. The factors contributing to these differences include a smaller sample size (29
versus 52), a new set of observers, different animals, and an additional hole board that was
handmade. Future studies should try to maximize their likelihood of observing significant results
by having a sufficiently large sample size (minimum of 25 rats per neonatal group), reducing the
number of observers to ensure consistency across observations, using identical apparatuses, and
increasing the amount of bedding used in the open field test to optimize the possibility of
marbles being buried.
All in all, our study did confirm the face validity of the neoclomipramine model for OCD
behavior as assessed by marble burying. The present study also contributed to the predictive
validity of the behavioral assays used to characterize the model, as demonstrated by the
significant differences between drugs collapsed across the rat condition for the total number of
marbles checked, poke/hole ratio, and the percentage of pokes in the first half of the trial.
Previous research has shown that abnormalities within the cortico-striatal-thalamic
circuits, comorbidity with anxiety disorders/mood disorders/impulse-control/substance abuse
disorders, and moderate heritability all contribute to the prevalence and debilitating effects of
OCD, and thus these are all areas in which more research would be helpful to further tease out
the biological causes of OCD (Greenberg et al., 2003). In addition, it would be helpful in the
33

future to examine the neurophysiology of neoclomipramine and neosaline rats to determine how
they respond differently to various drugs that have known effects in humans with OCD, so as to
further our understanding of the neurochemical basis of the disorder.
34

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