Evaluation of Dopaminergic

Neuron Loss in 6-OHDA Lesion

of Nigrostriatal Pathway in Rats
Tyler Mosdell
Neuroscience Discipline, The University of Western Australia, 35 Stirling
Highway, Crawley WA 6009, Australia

1 INTRODUCTION
Apoptotic dopaminergic neuron depletion is most commonly studied in cases of
late stage Parkinsons disease. Whilst the process of depletion is well understood,
the cause of this rapid neuronal loss still eludes us. The loss of dopamine
producing neurons in Parkinsons commonly occurs in the nigrostriatal pathway
in the brain. This pathway extends as an efferent connection from the substantia
niagra to the corpus striatum (Deumens, Blokland & Prickaerts, 2002; Vallone,
Picetti & Borrelli, 2000). Though depletion in this area is commonly associated
with Parkinson-like symptoms, links have been made to other neurological
disorders such as schizophrenia (Swerdlow & Koob, 1987). The most commonly
referred to symptom of Parkinsons is an impairment or dissociation of
sensorimotor response, it is these primary symptoms which have been linked to
the dopaminergic cell loss in this area of the brain (Nieoullon & Coquerel, 2003;
Mansbach, Geyer & Braff, 1988). Being one of the major dopamine pathways in
the brain, the use of homologous animal models has proven to hold clinical value
in behavioural lesion studies (Deumens, Blokland & Prickaerts, 2002).
The chemical 6-hydroxydopamine or 6-OHDA, is commonly used in such studies
due to its selective neurotoxicity towards dopaminergic and noradrenergic
neurons (Zigmoid, Abercrombie, Berger, Grace & Stricker, 1990). Pre-treating
subjects with desimipramine is common to prevent lesioning of noradrenergic
neurons (Espejo & Minano, 1999). By locally introducing this chemical to the
substantia niagra pars compacta, cell loss typical of Parkinsons, can be
mimicked, and physiological and behavioural responses can be analysed. The
call for this approach to Parkinsons research was made due to the fact that

symptoms of the disease only become evident once dopaminergic neuron

depletion is in excess of approximately 80% at which point the effectiveness of
treatment plans has been severely decreased (Robinson & Whishaw, 1988).
However, that is not to say that Parkinsons does not affect the brain at earlier
stages, in fact the changes occurring in the brain are greatest during the earlier
stages and if not for severe neural compensatory responses, symptoms would
have a much earlier onset. Early presynaptic compensatory responses, such as
an increase in the synthesis, release and uptake of dopamine, are able to buffer
the reduction in dopaminergic neurons leaving the disease undetectable in terms
of behavioural symptoms (Stachowlak, Keller, Stricker & Zigmond, 1987;
Zigmond, Abercrombie, Berger, Grace & Stricker, 1990). Compensatory
responses are present not only in these early stages but through to cell losses in
excess of 90%. Increased receptor density as well as super-sensitivity continue to
reduce the effects of the cell loss. The existence of such compensatory
responses is supported in; studies on homologous animal models showing an
increase in D2 dopamine receptor density and supersensitivity in cases of full
lesion (Kostrzewa, 1995; Creese, Burt & Snyder, 1977; Gerfen, Miyachi, Paletzki &
Brown, 2002), as well as post-mortem patient studies indicating an increase in
binding to both D1 and D2 dopamine receptors (Deumens, Blokland & Prickaerts,
2002). By buffering this decrease in dopaminergic neurons through both pre and
post synaptic methods, the brain is able to maintain a degree of normal function.
Dopamine agonists, primarily amphetamine, are often used as to highlight the
extent to which dopaminergic cell asymmetries exist in the brain. The response
to this class of drugs can be directly correlated to the percentage of
dopaminergic neuron loss. Amphetamine acts on dopaminergic cell vesicles,
causing them to release dopamine, as well as on presynaptic dopamine
transporters, causing them to reverse direction and pump dopamine into the
synapse (Schwarting & Huston, 1996; Jones, Gainetdinov, Wightman & Caron,
1998). Due to the presynaptic action of this drug, some presynaptic neurons
must remain (Hudson et al., 1993). This is primarily seen in low to mid-range
neuron depletion and so amphetamine is most effective as an indicator at this
range. In higher levels of neural depletion, apomorphine is typically used as it
stimulates the post-synaptic receptors. This has a much greater effect in near
complete neuron loss due to supersensitivity. Because apomorphine is acting on

supersensitivity in high levels of cell loss, it will often invoke high levels of
contralateral activity in the brain (Hudson et al., 1993, Schwarting & Huston,
1996).
In homologous animal 6-OHDA lesion studies, amphetamine introduction induces
ipsilateral turning due to the increased activity of remaining dopaminergic
neurons on the unlesioned side. The number of turns per minute is proportional
to the relative numbers of dopaminergic neurons on each side of the brain
(Deumens, Blokland & Prickaerts, 2002). Conversely apomorphine, invokes
contralateral turning due to the increased activity, brought on by
supersensitivity, of dopaminergic neurons on the lesioned side. The number of
turns per minute, in this case, seems to depend primarily on the presence this
supersensitiy, as in partial lesions, effects of the drug are negligible (Hudson et
al., 1993). By comparing the effects to each drug, an estimate for the
percentage of cell loss can be made. It is this concept which is used in animal
models to determine the extent of lesioned brains and thereby assess the effects
of a particular percentage of cell loss.
The use of homologous animal models is a viable way of extrapolating
information about the disorder through from early stages to late onset
symptoms. It aids the understanding of compensatory feedback systems which
seem to mask the symptoms of the disorder in early stages. By unilaterally
lesioning dopaminergic neurons in the substantia nigra pars compacta, we can
assess the response to both amphetamine and apomorphine and thereby
correlate behaviours of each subject with the percentage of dopaminergic cell
loss.

2 MATERIALS AND METHODS

2.1 SUBJECTS
Male Sprague-Dawley albino rats were used as subjects in both recovery and
non-recovery procedures in this experiment (N=36). Upon ethics approval, rats
were sourced by UWA from Animal Resource Centre and care was taken to
follow University Animal Experimentation Ethics Committee guidelines. Rats had
an average weight and standard deviation of 403.33g (28.88g) for non-recovery

group and giving an estimated age of 11 weeks, and 434.50g (34.88g) for
recovery group giving an estimated age of 12 weeks (Animal Resources Centre,
2014). All instruments and surgical preparations were supplied by UWA Shenton

Park Animal Research Facility. Drugs used were prepared by qualified staff prior
to procedures.

2.2 BEHAVIOURAL TESTS

2.2.1 Testing Procedure
Rats underwent behavioural testing both pre and post operatively. Initial tests
were used to determine the presence or extent of any pre-existing neural
asymmetries. All tests were performed initially under baseline conditions.
Locomotive test was repeated 20 minutes after subjects were given 0.3mg/kg
apomorphine via intra-peritoneal injection and 40 minutes after subjects were
given 2mg/kg amphetamine via intra-peritoneal injection. Baseline and
apomorphine locomotive, as well as sensorimotor tests, were repeated 2 weeks
post-surgery, whilst amphetamine locomotive test was repeated 3 weeks postsurgery. Subjects were graded on stereotypy at each locomotive test with a scale
ranging from 0 to 5. Testing criteria was as follows; 0 = asleep (eyes closed, lying
down, no movement), 1 = awake, minimum movement, some grooming, 2 =
mostly still but occasional bouts of locomotion and/or sniffing, 3 = intermittent
bouts of locomotion and/or sniffing, 4 = continuous locomotion and/or sniffing,
maybe some occasional bouts of licking with snout contact with cage surface, 5
= continuous sniffing with bouts of licking mostly in one spot and continuous
snout contact.
2.2.2 Locomotive Test
Subjects were placed in a cylindrical testing enclosure and observed over a
period of 15 minutes with the number of clockwise and anticlockwise turns being
recorded.
2.2.3 Sensorimotor Tests
Subjects were placed atop an elevated circular testing platform and randomly
touched with a neuropathy probe on either the left or right flank, repeated 5
times for each side. The test was repeated with the whiskers. The number of

responses to the stimuli, defined as head or body movement towards the stimuli,
was recorded.
Subjects were placed atop an elevated circular testing platform and a square of
adhesive tape, roughly equal to the size of the extended paw, was applied to one
of the forepaws. The time taken to remove the applied tape was recorded for
each of three trials per paw and averaged.

2.3 OPERATIVE PROCEDURE

All subjects were given; 0.16mg/kg atropine via subcutaneous abdominal
injection as an anticholinergic, and 5mg/kg carprofen as a NSAID (Non-Steroidal
Anti-Inflammatory Drug). Subjects were then given 1.5mL/kg anaesthetic solution
(3:2:1 ketamine, medetomadine and saline respectively) approximately 10
minutes prior to operative procedures. Hind limb and palpebral reflexes were
tested throughout procedure at 15 minute intervals with the drug having an
expected effective period of 45 minutes. Re-dosing of 0.75mL/kg anaesthetic
solution was administered based on these tests.
Subjects were prepared by shaving the head and applying sterilising solution.
They were then secured in stereotaxic apparatus (Stoelting Stellar apparatus). A
mid-sagittal, rostro-caudal incision was made extending from 1mm behind the
eyes to 2mm before the end of the skull. Connective tissue was detached using a
blunt blade and skull was exposed. Tissue was secured back using a repurposed
paperclip. Compression gauze was used to cease any localised bleeding. With
lambda and bregma skull landmarks visible, care was taken to level the skull
prior to operative procedures. Bregma coordinates were used to adjust atlas
coordinates for substantia nigra pars compacta (Paxinos & Watson 1997; Table
1). A burr hole was made at the AP, ML coordinate intersection using a small
Dremel rotary tool. A cannula was slowly lowered to the DV coordinate and 8L
of solution (10mM 6-OHDA and i=0.01% ascorbic acid) was injected at a rate of
1L/min. Cannula was slowly removed after leaving to rest for one minute.
Table 1; Adjusted atlas coordinates. Atlas coordinates were relative to bregma and so bregma
coordinates were subtracted to give adjusted coordinates.

Coordinate

Bregma

Atlas

Adjusted

AP

5.4

5.0

0.4

ML
DV

0.5
-4.9

2.1
7.9

-1.6
3.0

Incision was swabbed and closed with surgical clips. Once secure, lidocaine
(i=1%) was injected into the surrounding tissue as a local anaesthetic. Subjects
were given 0.1mg/kg atipamezole via intra-peritoneal injection as an alpha2adrenergic antagonist, to reverse sedative and analgesic effects. They were
moved to a heating mat and monitored until such time that they were able to
hold a position of sternal recumbency and demonstrate a swallow reflex.
Non recovery surgery followed the same surgical procedures one week prior to
the recovery surgery with the following exceptions: 6L methyl blue was infused
via cannula in substitute for 6-OHDA. Lethal dose of anaesthetic solution was
given post-surgery. Subjects brains were removed post-surgery and prepared as
brain slices to assess cannula placement.

3 RESULTS
3.1 LOCOMOTIVE RESPONSE RESULTS
Specimens 1 and 12 exhibited the only significant responses to apomorphine,
where significant is defined as values exceeding the cut-off specified by Arnt &
Hyttel (1984). All other subjects indicated no significant turning bias with
apomorphine treatment. Subjects 10, 15 and 12 were the only to exceed the
threshold for significant amphetamine response as specified by Creese, Burt &
Snyder (1977). Other subjects showed varying degrees of increased postoperative ipsilateral turning bias under amphetamine treatment. Subjects 14 and
17, conversely, showed an increased post-operative contralateral turning bias
under amphetamine treatment.
15.00

10.00
Apomorphine
5.00
Amphetamine
0.00

Turns per Minute (TPM)

-5.00
Cutoff for Significant Apomorphine Response
-10.00

Cutoff
for Significant Amphetamine Response
-15.00
14 17 13 18 8 16 5 6 3 11 7 4 2 10 15 1 12

Rat Number

Figure 1; Graph of locomotive response to apomorphine and amphetamine in terms of change in

turns per minute. Data was adjusted using the following difference equation: (I C) POST (I C)PRE
where I and C refer to ipsilateral and contralateral turns respectively and subscripts POST and
PRE refer to post-operative and pre-operative results respectively. Cut-offs for significant
responses obtained from prior studies (REF).

3.2 SENSORIMOTOR RESPONSE RESULTS

Sensorimotor response tests showed little correlation between tests as well as to
locomotive results. Subjects 14, 8 and 6 showing the only significant correlation
between the two sensorimotor tests. Subjects 17, 3 and 7 showed a strong bias
towards contralateral response in the tape removal test whilst subject 8 was the
only to elicit a strong bias in the touch response test, with it being towards
ipsilateral response.
100

-200
-150

50

-100
-50

Percentage Touch Response (%)

0
50

-50

100

Tape Removal Time (s)

150
-100

200
250

-150

300

Rat Number
Touches

Pad Tape

Figure 2; Graph of change in subjects response to asymmetric stimuli; percentage of responses to

flank and whisker touches with neuropathy probe from 10 touches, and average time taken to
remove adhesive tape from forepaw. Data was adjusted using the following difference equation: (IC)POST-(I-C)PRE where I and C refer to ipsilateral and contralateral turns respectively and subscripts
POST and PRE refer to post-operative and pre-operative results respectively. Cut-offs for
significant responses obtained from prior studies (REF). It should be noted that the axis for tape
removal time was reversed, as a decrease in time indicates an increase in response.

3.3 STEREOTYPY RESULTS

Stereotypy scaling was performed during baseline state, as well as after
apomorphine and amphetamine introduction, both pre and post-operatively. All
lesions showed increased or maintained stereotypy, post-operative, in all
conditions. Partial lesions showed an increase in stereotypy with amphetamine
treatment, and a decrease with apomorphine both pre and post-operative. The

same was observed for pre-operative stereotypy in full lesions. Post-operative

results for full lesions, however, showed increases in stereotypy for both
apomorphine and amphetamine. Figure 3 shows results for partial lesion and full
lesion groups. Locomotive results were used, in accordance with prior research,
to group subjects into these groups. Rats exhibiting locomotive results in excess
of 5 contralateral turns in with amphetamine as well as results in excess of 3
ipsilateral turns per minute with apomorphine were classes as full lesions for the
purpose of data grouping.
6
5
4
3

Average Stereotypy

2
1

Pre-Operative
Baseline

Apomorphine

Post-Operative

Partial Lesion

Amphetamine

Figure 3; Measure of average stereotypy in subjects Pre and Post-Operative under baseline,
apomorphine and amphetamine conditions. Data was separated into full (N=2) and partial (N=15)
lesions in subjects as prior research indicates paradoxical reaction to apomorphine in full lesions
(Creese, Burt & Snyder, 1977). All results recorded and calculated to 2 significant figures.

4 DISCUSSION
4.1 LOCOMOTIVE RESPONSE
To assess the effect of subjects lesioning on their turning response, the difference
between their baseline response and their amphetamine or apomorphine, was
used to ensure turning behaviour was as a result of the lesioning and not a
natural asymmetry in the brain (Deumens, Blokland & Prickaerts, 2002).
4.1.1 Full Lesions
Full lesions were defined as subjects exhibiting locomotive results for both
amphetamine and apomorphine greater than 6 contralateral turns and 3

ipsilateral turns respectively, as per prior research findings (Creese, Burt &
Snyder, 1977, Hefti et al., 1980, Carman, Gage & Shults, 1991, Arnt & Hyttel).
Based on this, subjects 1 and 12 were classed as having in excess of 90%
depletion. Full lesions exhibited the expected increase in ipsilateral turning with
amphetamine, whilst apomorphine induced increased contralateral turning. Due
to the almost complete loss of dopaminergic neurons, amphetamines effect on
the pre-synaptic terminals is negligible on the lesioned side, but causes high
concentrations of dopamine on the unlesioned side resulting in contralateral
turning. Apomorphines effect, however, receptor supersensitivity and its ability
to out-perform the normal cognitive response of the unlesioned side. It acts as a
direct post-synaptic agonist, resulting in ipsilateral turning (Hudson et al., 1993).
4.1.2 Partial Lesions
Partial lesions were subdivided into two additional groups, those exhibiting an
increase in ipsilateral turning response, greater than 3 turns per minute, to
amphetamine were classed as having 50-90% depletion. Subjects which did not
display this were classed as having lesioning of <50%, at which point
compensatory systems would have adequately compensated for the loss. Based
on this criteria, subjects 10 and 15 were classed as having depletion of 50-90%
whilst all other subjects had depletion of less than 50%. Partial lesions were
differentiated from full lesions, primarily, by their lack of response to
apomorphine. As there were still a large proportion of dopaminergic neurons
present on the lesioned side, supersensitivity did not develop. Due to the drugs
method of action, without supersensitivity neural asymmetries would not be
reflected with turning asymmetry.

4.2 SENSORIMOTOR RESPONSE

Dunnett et al. found that subjects exhibited a decreased response to stimuli
contralateral to their lesion. Increased lesioning appeared to correlate with the
extent of the deficiency. This trend was expected to be seen in subjects, postoperative. However, little correlation was found between responsiveness and the
extent of lesioning in this study. This lack of relationship can be expected in
partial lesioning due to sensorimotor recovery observed in previous studies
(Dunnett et al., 1981, Ljungberg & Ungerstedt, 1976). In this study, full lesions,

however, did not present any more sensory neglect than partial lesions and so
this lack of relationship is more likely due to subjective measuring methods. This
is again supported by the fact that even between the two sensory tests
performed, strong correlation was not found.

4.3 STEREOTYPY
Both partial and full lesion groups showed an increase in baseline stereotypic
response post-operatively. This has been commonly observed in studies and is
most likely due to stress induced dopamine release (Abercrombie et al. 1989).
Full lesions showed a post-operative increase with amphetamine whilst partial
lesions showed no significant increase. This suggest that the difference is most
likely attributable to receptor supersensitivity, as this mechanism is present in
the former but not latter. Apomorphine, however, gave a general decrease in
stereotypy relative to baseline pre-operatively, though it gave the most
differentiated response between partial and full lesions post-operative. Partial
lesions showed a slight increase, of 0.4 points, and full lesions showing
significant increase, of 4 points. This is, again, likely due to receptor
supersensitivity.

4.4 LIMITATIONS

AND

CONCLUSION

This study suffered from two main problems; the first of which was in the
subjective manner stereotypy and sensorimotor response were recorded. An
improvement to this could be to redefine the criteria for the tape test as time
until the first response to the presence of the tape is noted rather than time
taken for removal. This could offer more accurate representation of any
sensorimotor deficits as it has been found that said deficits could in fact impair
the subjects ability to remove the tape, thus effecting results. Additionally, more
quantifiable criteria could be used for stereotypy scaling.
In conclusion; by using apomorphine and amphetamine responses, an estimation
of the extent of each lesion was able to be made. Amphetamine was effective at
scaling partial lesions as well as indicating subjects with very little lesioning.
Apomorphine on the other hand, was highly effective at indicating subjects with
high levels of lesioning, typically in excess of 90%. Behavioural responses

correlated to compensatory methods present in the brain to supress the effects

of dopamine depletion. Ultimately, all variations in behaviour were related to the
level of depletion present in each subject and supported theories developed in
prior studies.

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