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Apomorphine Induces Contralateral Rotation
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Running Head: APOMORPHINE INDUCES CONTRALATERAL ROTATION
Apomorphine Induces Contralateral Rotation in Rats with Unilateral 6-hydroxydopamine
Lesions of the Substantia Nigra
Andrew Peterson
Davidson College, Davidson, NC
Word Count:
Abstract- 119 words
Introduction- 486 words
Discussion- 994 words
In partial fulfillment of the requirements for PSY303
Dr. Julio Ramirez
12/11/2015
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Abstract
Parkinson’s disease (PD) is characterized by a loss of dopaminergic neurons in the substantia
nigra (SN), resulting in alterations in activity of the basal ganglia, which is responsible for the
motor deficits associated with PD. Lesions of the nigrostriatal pathway with 6hydroxydopamine (6-OHDA) have been used as successful models for PD; one characteristic of
this model is rotational behavior either towards or away from the lesioned side depending on the
dopamine agonist administered. We used the rotational behavior of this model to determine the
identity of a mystery substance administered to rats lesioned in the nigrostriatal pathway with 6OHDA. Injection of the “mystery substance” resulted in vigorous contralateral rotations;
identifying the unknown dopamine agonist as apomorphine.
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“Mystery Substance” Induces Contralateral Rotation in Rats with Unilateral Lesions of the
Substantia Nigra Using 6-hydroxydopamine
Parkinson’s disease (PD) is the most common neurodegenerative movement disorder;
affecting more than 0.1% of the population older than 40 years of age (Dawson & Dawson,
2003). Patients present with slowness of movement, rest tremors, rigidity, balance issues, and
some also suffer from anxiety, depression, autonomic disturbances, and dementia (Wichmann,
Vitek, & DeLong, 1995). Neurologically PD is characterized by the loss of nigrostriatal
dopaminergic neurons and the presence of “Lewy Bodies” (LBs) in various types of neurons.
Animal models for PD are used to mimic the dopaminergic cell loss and the behavioral
deficits seen in human PD patients. 6-hydroxydopamine (6-OHDA) was one of the first agents
used in PD models to destroy dopaminergic neurons in the substantia nigra (SN) (Deumans et al.,
2002, Beal, 2001). After 6-OHDA is injected into the SN, it selectively accumulates in the
dopaminergic neurons, killing them by damaging the mitochondria and allowing toxic ⍺synuclein to accumulate, and it has minimal negative effects on the nearby GABA neurons. This
is the same damage that is seen in human PD patients (Wichmann, Vitek, & DeLong, 1995;
Zeevalk et al., 1996).
Bilateral and unilateral lesions have been done on this model. While bilateral lesions are
more representative of the conditions seen in human PD patients, unilateral lesions offer up
unique experimental opportunities (Ungerstedt, 1971a,b). Unilateral lesions with 6-OHDA allow
one hemisphere to serve as an internal control and it offers the opportunity to study drug-induced
changes in DA availability (Beal, 2001). The direction of rotational behavior induced by the
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administration of drugs in rats with unilateral lesions of the SN corresponds to their mechanisms
of action at the dopaminergic synapsis (Betarbet et atl, 2002; Casas et al., 1988). When
dopamine concentrations are increased in the synaptic cleft, ipsilateral turning is demonstrated.
Stimulation of supersensitive dopaminergic receptors in the denervated-striatum induces
contralateral rotational behavior (Herrera-Marschitz, 1986). Apomorphine and amphetamine are
two commonly used drugs to induce rotation in this model. Ungerstedt (1971a,b) used both in
his unilateral 6-OHDA lesioned rats and found that amphetamine causes DA to be released from
undamaged neurons and apomorphine produces activity in DA receptors primarily on the lesion
side. Overall, he demonstrated that tats will turn toward the side with the least amount of
dopamine activity. When challenged with dopamine agonists, the specific rotational behavior of
this model can help determine the neurological effects of the agonist on the synapse.
This experiment used rotational behavior to determine the identity of a “mystery
substance,” either apomorphine or d-amphetamine, injected into rats with unilateral 6-OHDA
lesions of the SN. The model initially described by Ungerstedt has been repeatedly tested and
will allow us to accurately predicting the identity of the mystery substance based on the
rotational behavior observed. If contralateral rotation is exhibited after surgery, the identity of
the mystery substance is apomorphine; if ipsilateral rotational behavior is observed, it is damphetamine.
Methods
Subject
Subjects included 18 male Sprague-Dawley rats (Hilltop Lab Animals, Scottsdale, PA)
that initially weighed between 325-375 grams. The rats were individually stored in the animal
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facility in 45cm x 24cm x 21cm wire cages with ad libitum access to dry food and water at all
times until deprivation 24 hours before the surgery. All rats were given regular and identical
enrichment throughout the experiment. If the weight of a rat dropped below the target weight,
“wetmash” was provided. The rat colony was on a 12 hour light/dark cycle (7:00 am on, 7:00
pm off), and the temperature of the colony remained at a constant 21.1 ± 1.1℃. The experiment
conformed to guidelines set by the National Institute of Health and approved by the Davidson
College Animal Care and Use Committee.
Apparatus
Behavioral testing was done in a Roto-Rat Test Station (Columbus Instruments,
Columbus, OH) connected to a Dell- Optiplex Gx620 computer with an Intel processor running
the SOF-801 Roto-Rat System version 2.02 made by Med. Associates, Inc. The metal bowl has a
diameter of 46.5 cm, a depth of 15.1 cm. The plexiglass cylinder on top of the metal bowl had a
height of 29.4 cm. A velcro harness is attached to a long metal crossbar at the top of the
plexiglass wall by a 37.9 cm flexible steel wire. The apparatus detects partial (180º) and
complete (360º) clockwise (ipsiversive) and counterclockwise (contraversive) rotations through
this steel wire.
Design
This study followed a pre/post design. The rats underwent seven days of pre-operational
testing one week prior to their surgery and seven days of post-operational testing two weeks after
surgery. The independent variable in this experiment is the “mystery substance”, either
apomorphine or d-amphetamine, given to stimulate dopaminergic synapses and induce rotational
behavior. The dependent variable was the direction of the rotational behavior, either ipsiversive
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or contraversive, induced in the rats following injection of the mystery substance. In this
experiment, the independent variable (the identity of the mystery substance) was the same for all
test subjects. Only one test group was used in this experiment; all subjects went through the pre/
post behavioral testing and received injections of the mystery substance. The unilateral lesions
served as an internal control for this experiment and allowed us to determine the identity of the
mystery substance.
Procedure
Behavioral Testing: Seven days before the beginning of behavioral testing, approximately
three weeks before surgery, rats were handled daily for at least 10 minutes to accustom them to
human contact and handling. Pre-surgery behavioral testing began seven days before the 6OHDA lesion surgery; rats were given intraperitoneal injections of 0.5 ml/kg of the mystery
substance. After at least 10 minutes, each rats rotational behavior was recorded in the Roto-Rat
Test Station over a 10 minute period.
Post-surgery behavioral testing was conducted following the same procedure as presurgical testing for a period of seven days exactly two weeks after surgery.
Surgery: Surgical lesioning of the right SN with 6-OHDA was performed on all 18
subjects. The rats were anesthetized with an intraperitoneal injection of Ketamine (1 ml/kg) and
Xylazine (0.4 ml/kg). Intraperitoneal injections of supplemental anesthesia were given
throughout surgery if necessary: half of the original ketamine injection and 0.07ml Xylazine. An
incision was made above the target area, and measurements were taken relative to bregma
according to the rat’s weight (anterior/posterior: <300g, -3.0mm; 300-400g, -3.2mm; >400g,
-3.5mm; medial lateral: -1.8mm). After the injection site is determined, a craniotomy is done
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and a 5µl Hamilton syringe is lowered relative to the dura and according to weight (<200g,
-8.2mm; 200-300g, -8.3mm; >300g, -8.4mm). We allowed the cannula to sit in the brain for 2
minutes before injecting 4.0µl of 6-OHDA (2µg/ml 6-OHDA in 0.9% saline vehicle with 0.02%
ascorbate, Sigma Chemical Co.) at a rate of approximately 0.5µl/min. After injection, the
cannula remained in the brain for an additional 5 minutes to avoid unintended damage from
displacement of brain matter before being slowly removed over a 1 minute period.
Histology: Within 24 hours after post-surgical behavioral testing was completed, the rats
were euthanized with an overdose of Ketamine (0.7-0.8 cc), xylazine (0.3-0.4 cc), and placed in
a CO2 chamber before they were transcardially perfused with 10% neutral-buffered formalin.
After perfusion, the rats were decapitated and the brains were removed for preservation in 10%
neutral buffered formalin for three days then transferred to a sucrose formalin solution until
slicing. A microtome (Scientific Instruments) was used to coronally slice the brain into 40µm
thick sections. Slices were mounted on pre-soaked gelatin glass slides and stained with a cresol
violet acetate stain in order to microscopically analyze the brain tissue for the extent of the
lesion.
Results
Histology
Analysis of the brain tissue of the rats that rotated demonstrated extensive destruction to
the SN region. See Fig. 1 for a representation of a partial lesion of the SN with 6-OHDA that
resulted in contraversive rotation. The area shaded in black in the lower right portion of the
diagram is the 6-OHDA lesion, and the narrow shaded rectangle in the upper right denotes the
cannula track. Analysis of brain tissue from the 6 non-rotating rats was revealed that the 6-
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OHDA lesion was off-target and did not significantly damage the SN, which is why they did not
exhibit the desired rotational behavior. These 6 non-rotating rats were excluded from statistical
analysis because they did not have extensive enough lesions to serve as effective PD models and
to identify the mystery substance.
Rotational Behavioral
Rotational behavior from the 12 subjects that rotated was averaged to generate a list of
mean values for pre-surgery contraversive rotations (Pre-Contra), pre-surgery ipsiversive
rotations (Pre-Ipsi), post-surgery contraversive rotations(Post-Contra), and post-surgery
ipsiversive rotations(Post-Ipsi). A two-tailed, paired t-tests was performed on the data and a pvalue of 0.05 was considered significant.
Figure 2 demonstrates rats did not significantly favor the contraversive or ipsiversive side
before surgery (t(12) = 0.736, p = 0.477); on average, they did not complete many full
contraversive (m = 0.514) or ipsiversive (m=0.609) rotations when injected with the mystery
substance before surgery. After injection of the mystery substance, there was a significant
difference between ipsiversive and contraversive rotation. Figure 3 indicates that post-surgery
ipsiversive rotations were significantly lower than pre-surgery ipsiversive rotations (t(12) = 3.43,
p = 0.00559); Figure 4 demonstrates that post-surgery contraversive rotations were significantly
higher than pre-surgery contraversive rotations (t(12) = -4.47, p = 0.000954). Figure 5 shows
there were significantly more contraversive rotations than ipsiversive rotations after surgery
(t(12) = 4.484, p = 0.000926); in rats with a unilateral lesion of the SN, the mystery substance
clearly produced vigorous contraversive rotation.
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Discussion
Injection of the mystery substance following 6-OHDA lesions of the SN significantly
increased contraversive rotation, and there was no significant change seen in ipsiversive rotation.
According to previous studies, vigorous contraversive rotations in rats following injection of a
dopamine agonists in rats unilaterally lesioned with 6-OHDA is induced by apomorphine
(Ungerstedt, 1971a,b). Destruction of the dopaminergic neurons in the SN lowers the amount of
DA reaching the postsynaptic cells and stimulates the development of supersensitivity in DA
receptors in the lesioned side (Ungerstedt, 1971b, 1975). Apomorphine is an agonist for the
dopamine receptor while amphetamine increases the release dopamine from the synapse (Anden
1967); therefore, apomorphine will have the greatest effect in rats with supersensitive dopamine
receptors on the lesioned side (Ungerstedt, 1976). Amphetamine, as a dopamine-releasing agent,
would have the greatest effect in dopamine synapses with surviving dopaminergic neurons
(Carman, 1991). However, since 6-OHDA selectively accumulates in the dopaminergic
neurons, killing the neurons by damaging the mitochondria (Dawson & Dawson, 2003), only the
dopamine neurons will be affected by this lesion, not the receptors in the striatum. Rats will turn
away from the side of the brain with the most dopaminergic activity, so they contraversively
rotate when injected with drugs that act as post-synaptic dopamine agonist, like amphetamine
(Hefti, 1980).
Apomorphine and D-amphetamine both produce rotational behavior in rat models
unilaterally lesioned with 6-OHDA. Previous research indicates that the extent of the SN lesion
influences the rotational behavior observed. Hudson and et al (1993) found animals with
extensive lesions (>90% depletion of dopamine in the striatum) rotated substantially after
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injections of apomorphine because supersensitivity of stratal postsynaptic receptors would
develop. When less than 90% of the nigrostriatal neurons were destroyed, no contraversive
rotation was observed because no supersensitivity of postsynaptic receptors had developed for a
dopamine agonists like apomorphine to act upon (Hudson, 1993; Hefti, 1980). In contrast,
amphetamine induced rotational behavior in animals when only 50% of neurons in the SN were
destroyed (Hefti, 1980). Additionally, lesions that were depleted DA levels by 70-90% also
rotated when challenged with d-amphetamine but not apomorphine. Therefore, we can be certain
that all of rats included in the statistical analysis had extensive lesions of the SN because they
rotated in response to apomorphine. It is possible that some of the rats that did not rotate would
have rotated if given injections of amphetamine instead of apomorphine; their lesions of the SN
may not extensive enough to induce contraversive rotation when challenged with apomorphine.
This means that using apomorphine to select for successful lesions is limiting the analysis to
subjects with nearly complete loss of the dopaminergic neurons. However, this is a problem
when modeling PD because it is a progressive disease in humans, and testing on models that only
reflect the later stages of the disease could inhibit the discovery of potential treatments.
PD is a progressive disease in humans, and it is important to have animal models that can
mimic the progressive stages of the disease. In order to develop these models, methods for
detecting the extent of dopaminergic neuron loss are necessary (Sauer, 1994). Park et al (2015)
tried to model the progressive stages of PD by injecting a mouse model with different
concentrations of 6-OHDA into the right medial forebrain bundle. The four groups demonstrated
significant changes motor function, dopaminergic cell loss in the SN, and neuronal firing rates in
the striatum; thus mimicking each progressive stage of human PD because larger dosages that
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resulted in more extensive lesions resulted in more intense symptoms. These results demonstrate
that different sizes of lesions can produce behaviors associated with PD, even if the model’s do
not exhibit the expected rotational behavior.
While this experiment yielded clear results that are supported by previous research, the
design could have been easily improved if we had more time and resources. Using apomorphine
induced rotation to determine which subjects to include in the statistical analysis means that only
extensively lesioned models are being considered, which is only representative of late-stage PD.
A more effective way determine which subjects to include in the statistical analysis would be to
quantitatively assess the extent of the 6-OHDA lesion. Immunocytochemical analysis could be
done to reveal the number of tyrosine hydroxylate-positive neurons in the lesioned area. When
compared to the non-lesioned side, this would provide an quantitive means of assessing the
extent of neuron destruction from the lesion (Anaya-Martinez et al. 2006). Additionally,
including a control group that undergoes surgery and receives an injection of saline instead of 6OHDA would help determine if the turning behavior was a result of the lesioning rather than the
surgery itself. However, many previous studies have included such a control, and their results
reveal that injection of saline into the SN has a negligible behavioral and histological effect
(Barneoud, 1995; Anaya-Martinez et al. 2006).
The goal of this PD rat model is to gain a better understanding of Parkinson’s disease in
the hopes that we can find a cure. Previous research has demonstrated that this model is
continually being refined in order to better mirror the conditions seen in human PD patients.
While there are some limitations to the model we used in the experiment for modeling the stages
of PD, it does provide us an effective tool for identifying the “mystery substance.” Post-surgical
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rats exhibited contraversive rotational behavior when challenged with apomorphine, the mystery
substance, because apomorphine is a dopamine receptor agonists. The development of
supersensitive postsynaptic dopamine receptors in the lesioned side of the brain resulted in an
unequal activation of the dopaminergic pathway when stimulated with apomorphine, which is
behaviorally represented by contraversive rotation. These results are consists with previous
findings and further demonstrate the damaging effects of dopaminergic loss in the SN.
Hopefully continued study and refinement of this PD model will further increase our
understanding of the neurological mechanisms underlying PD in humans.
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Figure Captions
Figure 1. This diagram represents the location of a partial lesion of the substantia nigra of rat
326, who did rotate after surgery. The areas colored in black were destroyed by the lesion. The
damaged regions in the lower right portion of the diagram are the 6-OHDA lesion. The damaged
area in the upper right hemisphere represents part of the cannula track.
Figure 2. Pre-Surgery Contraversive vs. Pre-Surgery Ipsiversive. Injection of the mystery
substance before surgery did not result in a significant difference between the number of
contraversive rotations and ipsiversive rotations (p = 0.479). The bars represent the number of
rotations during a 10 minute session in the Roto-Count 8 System ± the standard error above the
mean.
Figure 3. Pre-Surgery Ipsiversive vs. Post-Surgery Ipsiversive. Injection of the mystery
substance resulted in a significantly fewer ipsiversive rotations after surgery (p = 0.006). The
bars represent the number of rotations during a 10 minute session in the Roto-Count 8 System ±
standard error above the mean.
Figure 4. Pre-Surgery Contraversive vs. Post-Surgery Contraversive. Injection of the mystery
substance resulted a significant difference in the number of contraversive rotations before and
after surgery (p = 0.001). The bars represent the number of rotations during a 10 minute session
in the Roto-Count 8 System ± standard error above the mean.
Figure 5. Post-Surgery Contraversive vs. Post-Surgery Ipsiversive. Injection of the mystery
substance after surgery resulted in a significant difference in the number of contraversive and
ipsiversive rotations (p = 0.001). The bars represent the number of rotations during a 10 minute
session in the Roto-Count 8 System ± the standard error above the mean.
Apomorphine Induces Contralateral Rotation
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Figures
Figure 1
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Figure 2
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Figure 3
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Figure 4
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Figure 5