exploring compensatory plasticity via dopamine depletion on rats and the effects of apomorphine on...
TRANSCRIPT
Running Head: EXPLORING COMPENSATORY PLASTICITY1
Exploring Compensatory Plasticity via Dopamine Depletion on Rats and the Effects of
Apomorphine on Unilateral Lesioning of the Striatum
Joshua Rochotte
Rutgers University
Running Head: EXPLORING COMPENSATORY PLASTICITY2
Abstract
Dopamine is an essential Neurotransmitter that functions in a multitude of ways but the research
being presented aims to focus on the motor specificity function of Dopamine. Specifically, when
rats are unilaterally lesioned with dopamine receptor antagonists, what effect will they have in
the coordination of fine motor movement and sensorimotor integration. Previous research has
shown significant results in successful unilateral dopamine depletion and investigators hope to
replicate previous findings. Parkinson’s disease is a debilitating neuromuscular degenerative
disease and by studying the rat models, light is hope to be shed on the chemical dynamics and
the physical manifestations of the diseases inner workings. Using apomophine to test the affinity
of the neurons will also be stressed. Findings show significant support to the hypothesis and
demonstrate a need for more investigation on the subject.
Running Head: EXPLORING COMPENSATORY PLASTICITY3
Introduction
Dopamine is an essential Neurotransmitter and works on multiple different pathways in
the CNS that coordinate movement and emotion. Dopamine is also involved in risk taking
behaviors and acquisition of and entailment of rewards. In the research being presented, the
movement pathways will be stressed, with special focus placed on neuropathology's resulting
from the loss of dopamine, in particular Parkinson's disease. The striatum is a mesencephalon
structure that is necessary for coordination of movement and in the controlling of fine motor
movement and sensorimotor integration in the body. It uses dopamine in two main receptor
types, known as D1 and D2 receptors. These two receptor subtypes stud themselves in the pre
and post synaptic membranes and are prevalent in a main motor pathway known simply as the
nigrostriatal and work opposite to each other (Sonsalla, 1998). The nigrostriatal pathway is
important in maintaining these motor behaviors and when dopamine is lost, these connections
become weaker and the pathway degrades. This pathway contains projections from the substantia
nigra pars compacta to the dorsal striatum, ergo the name. Parkinsons disease is a
neurodegenerative condition in which dopamine secreting neurons are degraded in a midbrain
area of the brain known as the substantia nigra. The condition causes motor movement
deprivation, loss of control of fine motor movement and it causes tremors(Schapira, 1999).
Previous research has documented the marked effects of dopamine on motor coordination
and successful functioning in time of deprivation in rats, and this is where the focus o the
literature will be. Unilateral dopamine depletion in a rat model is a good model for human
parkinsons disease because it models well the neglect in the brain on the neurons (Miklyaeva,
1994). In these experiments, researchers employed the use of a powerful Neurotoxin known as
six hydroxy-dopamine(6OHDA). This chemical acts on Dopamine receptors and shuts them off
Running Head: EXPLORING COMPENSATORY PLASTICITY4
by acting antagonistically, forcing a shut down of dopamine production and dopamine signal
transduction. Lesions in the studies have been performed unilaterally so that you can maintain an
intramodullar control of the subjects. By lesioning the one side with a 6OHDA injection to the
Striatum, one can effectively study the effects against a control in the same rat. Effects of this
Unilateral depletion have been shown to model a human parkinsonian patient well, with neglect
of the contralateral sense patterns and increased neural affinity on the ipsilateral side to the lesion
( Xu, 2005). What’s interesting is the rats fail to integrate sense on the contralateral side to the
lesion because of the crossing that occurs via sensorimotor integration. So when the left side is
lesioned, the right side losses sense in the rat models.
Another piece of this research is examining the effects of a dopamine receptor agonist
known as Apomorphine. This drug was chosen due to the effects it has on receptors for
dopamine. Apomorphine is a receptor agonist that activates D1 and D2 receptor subtypes. It is
thought to be a good therapeutic option for Parkinson's because it up regulates dopamine,
especially in damaged cortical areas. However, in the rats, DA agonists have been used two-fold:
one for a method of detection instead of autoradiography and secondly as an experimental
conclusory step to figuring out who the diseased rats brains compensate for the motor deficits
caused by deprivation of DA.
The research here seeks to confirm conclusions about unilateral depletion of dopamine
that countless scientists have previously put forward. Will unilateral dopamine depletion truly
cut off the sense contra laterally and will Apomorphine have any interesting effects on the
damaged brains of the Sprague Dawley Rat Subjects. If the conclusions previously made hold,
yes.
Running Head: EXPLORING COMPENSATORY PLASTICITY5
Methods
Eight male Sprague-Dawley rats were individually housed in their respective shoebox
cages. They received food and water ad liitum and received a 12 hour light cycle with lights
going on at seven in the morning and off at seven in the night. All drugs were obtained from
Sigma. Subjects were given general anesthesia of Ketamine-Xylanine (80mg/kg,12mg/kg) in an
intraperitonal injection (i.p.). Desipramine (15mg/kg) injections were given 30 minutes prior to
the administration of the 6OHDA to promote selectivity of the neurotoxin blocking only
dopamine receptors. Marcaine (20mg/kg) was subcutaneously injected to the rat as it was
cleaned and ready to be operated on. Rats were shaved and prepped for surgery. Using no touch
sterilization, rats were cut on the midline from medioccular position to right behind the ears.
Sterotaxed and locked in, the rats were then marked out for the surgery. Coordinates for
every rat varied but followed the same coordinate structure: 3.8mm posterior to Bregma, 1.7mm
Lateral to bregma and 9mm ventral to the skull. Once inside, using a Hamiltion syringe, 3ul of
4mg/1.5ml of a .02% ascorbic acid solution of 6OHDA was administered unilaterally into the
medial forebrain bundle, just above the striatum, over a ten minute period. The needle was then
slowly retracted so as to avoid cortical damage in the upper gray matter. Rats were given 5-7
weeks to rest and to allow the drugs to take full therapeutic effect. Upon week 7, rats were
injected with apomorphine after sufficient behavioral testing was done. Primarily, rats were
placed in 4’ x 4’ x 18” enclosures. They were then prodded with cotton tipped sticks and
assessed for degree of rotation toward the stick, scoring the rats from one to four on three touch
points: head, forelimb and trunk. After they were recorded, rats entered a conical tube and were
assessed as to the direction of the rotation and the amount of spins performed in a 30 minute
Running Head: EXPLORING COMPENSATORY PLASTICITY6
interval post injection with Apomorphine (.25mg/kg). Rats were returned to their cages and left
to live until required life ending procedures were performed.
Results
After receiving drug administration, rats rested for five weeks. Animals (N=8) were
then behaviorally tested under two criteria. Rats showed a contralateral response (1.7925 +/-
1.32952) that was lesser than ipsilateral responses (3.1250 +/- .83452) recorded on the head
(Mean +/- STD). A paired T test showed a significant difference (t(7)=-3.126, p<.05). Forelimbs
showed just the same type of response contra-laterally (1.7000 +/- 1.35013) as opposed to
the higher ipsilateral response (3.1250 +/- 0.83452) on the same area. A paired t test revealed
significant decrease in contralateral function (t(7)=-3.435, p<.05). The trunk had a decreased
response on the contralateral (1.0750 +/- 0.99535) and the Ipsilateral (2.8750 +/- 0.64807)
response in comparison to the other two stimuli spots. A paired T test revealed the
significance in higher ipsilateral trunk response than contralateral (t(7)=-4.687 p<.05).We had
a marked difference between observed rotations in the rats contra-laterally (106.2500 +/-
115.52211) and the significantly lower amount as was expected on the Ipsilateral side (3 +/-
2.39046). A paired T test revealed a significant finding in terms of the contralateral versus
ipsilateral rotations (t(7)=2.468, p<.05). This lead us to reject the null hypothesis in favor of the
presented predictions.
Running Head: EXPLORING COMPENSATORY PLASTICITY7
Discussion
The research conducted fell right into expected grounds. The first behavioral test with
the cotton swabs showed a marked difference between the ipsilateral and contralateral sides
of the lesion. This meant that indeed it could be concluded that sensorimotor integration had
been cut off on the left side, left of course being contralateral to the lesion. This was noted in
all cases on the rats and even so far as some rats were swiped down the whole body with a
cotton swab and yielded no reaction to it. However, in most cases on the right, the rat
instantly senesced and reacted to the cotton swab being there and went for it. Figure 1 shows
the scoring point system used and Figure 2 shows a summitry of the results for the mean of
the rat subjects (N=8). The rats all seemed to have taken the treatment, showing proof of the
unilateral neglect due to contralateral lesioning of the medial forebrain bundle and cutting the
nigrostriatal pathway off.
In examining the effects of apomorphine on the reduced dopaminergic pathway
neurons, a few interesting observations came about. Firstly, some rats ended up turning
ipsilaterally, that is, towards the lesioned side. These rats generally exhibited some
contralateral and ipsilateral movement, suggesting an incomplete treatment or a failure of the
drug. This means that either not enough dopamine neurons were dealt with in the initial
6OHDA treatment stages or that through some weird take-up method, dopamine neurons had
been replaced or reopened. It’s important to note that during testing, all rats received
relatively the same amount of apomorphine and so some of the larger rats may have reacted
less to the apomorphine than some of the smaller rats. In a few subjects though, the
experiment went without a hitch. They demonstrated proper sensory motor deficits and then,
Running Head: EXPLORING COMPENSATORY PLASTICITY8
once the apomorphine had been administered, began to spin almost uncontrollably, sending
their spin total over two hundred and fifty spins. These rats took the therapy and the drug
worked as expected. These rats rotated around their left foot. They kept it planted and pushed
with the right, moving contra laterally to their lesioned brain side. This is as expected due to
the interactions that apomorphine has in he brain of the affected rates. See figure 3.
Apomorphine acts as a receptor agonist and increases flow of the neurons. In the presence of
the 6OHDA, however, the dopamine neurons are far fewer, but they pick up an extra additive
effect. This is because the neurons develop a higher affinity for dopamine, meaning they
process more of it in a quicker time frame. So, while there are little receptors present, the
large affinity for dopamine allows more signals to be transducer and sent from the lesioned
side. This means that the contralateral side will be induced with more motive force and thus
induce a higher rotation on that side. The Rats ended up exhibiting some other behaviors such
as grooming and self bitting mid rotations. This research points to the quasi therapeutic effect
of apomorphine and how if applied therapeutically, one might be able to maintain periods of
normal functioning. The hypothesis was confirmed and results supported the findings. One
might wonder about the therapeutic use of the drug and look for alternative dopamine
replacers. Animals need strong motor systems so they can move and survive and when
damaged and degrading, fine motor control becomes difficult.
Running Head: EXPLORING COMPENSATORY PLASTICITY9
References
Miklyaeva, E., Castaiieda, E., & Whishaw, I. (n.d.). Skilled Reaching Deficits in Unilateral
Dopamine-depleted Rats: Impairments in Movement and Posture and
Compensatory Adjustments. The Journal of Neuroscience, 14(11), 7148-7158.
Schapira, A. (1999). Science, medicine, and the future: Parkinson's disease. Bmj, 311-314. doi:
10.1136
Sonasella, P., Manzino, L., & Heikkila, R. (1988). Interactions of Dl and D2 Dopamine
Receptors on the Ipsilateral vs. Contralateral Side in Rats With Unilateral
Lesions of the Dopaminergic Nigrostriatal Pathway. The Journal of Pharmacology and
Experimental Theraputics, 247(1), 180-185.
Xu, Z. C., Ling, G., Sahr, R. N., & Neal-Beliveau, B. S. (2005). Research report: Asymmetrical
changes of dopamine receptors in the striatum after unilateral dopamine
depletion. Brain Research, 1038163-170.doi:10.1016/j.brainres.2005.01.033
Running Head: EXPLORING COMPENSATORY PLASTICITY10
Figure 1.
Figure 1. Above is shown the objective scoring ranges for movement of the rests body during
the sensorimotor portion of the behavioral testing. scores ranged from 0-4.
Figure 2.
Running Head: EXPLORING COMPENSATORY PLASTICITY11
Figure 2. Mean comparison between the Touches given to the rat Contralateraly versus
ipsilaterally in all cases. Error bars represent SEM”, (indicates p < 0.05).
Figure 3.
Head Forelimb Trunk0
1
2
3
4
IpsilateralContralateral
Location of Touch
Aver
age
Scor
e of
Re-
spon
se to
Tou
ch S
timul
us
Running Head: EXPLORING COMPENSATORY PLASTICITY12
Figure 3. Mean Comparison between Contralateral versus Ipsilateral Rotations in rat (n=8)
subjects. Error bars represent SEM, indicates p < 0.05.
Contralateral Ipsilateral0
12
24
36
48
60
72
84
96
108
120
Rotation Direction relative to Lesion
Mea
n Ro
tatio
ns u
nder
Ap
omor
phin
e