Exploring Compensatory Plasticity via Dopamine Depletion on Rats and the Effects of Apomorphine on Unilateral Lesioning of the Striatum

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


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.


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


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.


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


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.


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,


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.


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


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.


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


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

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Exploring Compensatory Plasticity via Dopamine Depletion on Rats and the Effects of Apomorphine on Unilateral Lesioning of the Striatum