This document describes various in vivo and in vitro models used for screening potential antiparkinsonian drugs. Some key in vivo models mentioned include: 1) antagonism of tremors induced by tremorine/oxotremorine in mice, 2) use of the MPTP model in monkeys to induce Parkinson's disease-like symptoms, and 3) antagonism of sedation induced by reserpine in mice. Important in vitro/ex vivo models include measuring dopamine-stimulated adenylyl cyclase activity and radioligand binding studies for dopamine receptors. The document provides details on the procedures, evaluations, and modifications of these various screening models.

1. BY
MALLAPPA. SHALAVADI,
M-PHARM I,
HSK COLLEGE OF PHARMACY,
BAGALKOT.

2. INTRODUCTION
It is a progressive neurodegenerative disorder .
Their is a marked deficiency in the dopaminergic
innervation of the basal ganglia owing to
degeneration of neurones in the substantia nigra.
Enhancement of dopaminergic transmission restores
at least partially motor function.
The decrease in dopaminergic activity in the basal
ganglia results in a relative excess of cholinergic
influence.
Therefore, dopaminergic agonists, such as levodopa, a
precursor of dopamine, and cholinergic (muscarinic)
antagonists can be combined in the treatment of
Parkinson’s disease.

3. SCREENING MODELS
I. IN VIVO MODELS
a)Tremorine and oxotremorine antagonism
b)MPTP model in monkeys
c)Reserpine antagonism
d)Circling behavior in nigrostriatal lesioned
rats
e)Elevated body swing test
f)Skilled paw reaching in rats
g)Stepping test in rats

4. II. IN VITRO AND EX VIVO MODELS
a. Experiments using rat striatal slices
b. Dopamine stimulated Adenylyl Cyclase
activity
c. Radioligand binding studies for D1 and D2
Dopamine receptors
d. Dopamine release from synaptosomes

5. a. Tremorine and oxotremorine antagonism
–
 PURPOSE AND RATIONALE
 The muscarinic agonists tremorine and oxotremorine
induce parkinsonism-like signs such as tremor, ataxia,
spasticity, salivation, lacrimation and hypothermia.
 These signs are antagonized by anticholinergic drugs.
 PROCEDURE
 Groups of 6-10 male NMRI mice weighing 18–22 g
are used.
 They are dosed orally with the test compound or the
standard (5 mg/kg benzatropine mesilate) 1 h prior
the administration of 0.5 mg/kg oxotremorine s.c.

6. Rectal temperature is measured before
administration of the compound (basal value) and
1, 2 and 3 h after oxotremorine injection.
Tremor is scored after oxotremorine dosage in 10 s
observation periods every 15 min for 1 h.
Tremor Score
absent 0
slight 1
medium 2
severe 3

7. Salivation and lacrimation are scored 15 and 30 min
after oxotremorine injection.
absent 0
slight 1
medium 2
severe 3
 EVALUATION
Hypothermia
The differences of body temperature after 1, 2 and 3 h
versus basal values are summarized for each animal
in
the control group and the test groups.
The average values are compared statistically.

8. Tremor
The scores for all animals in each group at the 3
observation periods are summarized.
The numbers in the treated groups are expressed
as percentage of the number of the control group.
Salivation and lacrimation
The scores for both symptoms for all animals in
each
group are summarized at the 2 observation
periods.
The numbers in the treated groups are expressed
as percentage of the number of the control group.

9.  MODIFICATIONS OF THE METHOD
 Johnson et al. (1986)
Developed a procedure for quantifying whole-
body tremors in mice.
Displacement of a free floating platform by
animal movement created a change in resistance
across a strain gauge.
Administration of oxotremorine, 2.5 mg/kg, i.p,
produced numerous high-frequency, high-
intensity peaks within 5 min.

10. Clement and Dyck (1989)
Constructed and tested a tremor monitor that quantitates
soman- and oxotremorine-induced tremors.
The device consisted of a force transducer, from which a
plastic beaker was suspended containing a mouse.
The signal from the force transducer was fed into a tremor
monitor and quantitated using the Applecounter from
Columbus Instruments.
Coward et al. (1977)
Recommended N-carbamoyl-2-(2,6-
dichlorophenyl)acetamidine hydrochloride (LON-
1954), a tremorigenic agent, as alternative to
oxotremorine for the detection of anti-Parkinson
drugs

11. b. MPTP model in monkeys
 PURPOSE AND RATIONALE
N-MPTP (N-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine) has been shown to cause
symptoms of Parkinson’s disease in exposed
individuals.
When administered to primates this compound
causes a partial destruction of basal ganglia and a
syndrome that resembles Parkinson’s disease.

12. How MPTP induce parkinsonism?

13.  PROCEDURE
Burns et al. (1983) treated 8 adult rhesus monkeys
weighing 5–8 kg over a period of 5–8 days with
cumulative intravenous doses of N-methyl-4-phenyl-
1,2,3,6-tetrahydropyridine (N-MPTP) up to 10–18
mg/kg.
These animals showed a parkinsonism like disorder
(akinesia, rigidity, postural tremor, flexed posture,
eyelid closure, drooling)
 Which was reversed by the administration of L-dopa.
The pathological and biochemical changes produced
by N-MPTP are similar to the well established
changes in patients with Parkinsonism.

14.  EVALUATION
The severity of parkinsonian symptoms is rated by
trained observers
 movement (0: normal; 1: reduced; 2: sleepy)
checking movements (0: present; 1: reduced; 2:
absent)
attention and blinking (0: normal; 1: abnormal)
 posture (0: normal; 1: abnormal trunk; 2: abnormal
trunk and tail; 3: abnormal trunk, tail, and limbs; 4:
flexed posture)
balance and coordination (0: normal; 1: impaired; 2:
unstable; 4: falls)
reactions (0: normal; 1: reduced; 2: slow; 3: absent)
vocalizations (0: normal; 1: reduced; 2: absent).

15.  MODIFICATIONS OF THE METHOD
Kebabian et al. (1992) tested a selective D1 receptor
agonist with antiparkinsonian activity in MTPT
treated marmosets.
Domino and Sheng (1993) studied the relative
potency of some dopamine agonists with varying
selectivities for D1 and D2 receptors in MPTP-induced
hemiparkinsonian monkeys.
Asin et al. (1997) tested a selective D1 receptor agonist
in rats previously given unilateral 6
hydroxydopamine injections and in macaques
previously given unilateral, intracarotid infusions of
MPTP.

16. c. Reserpine antagonism
 PURPOSE AND RATIONALE
Reserpine induces depletion of central catecholamine
stores.
The sedative effect can be observed in mice shortly
after injection, followed by signs of eyelid ptosis,
hypokinesia, rigidity, catatonia, and immobility.
These phenomena can be antagonized by dopamine
aonists.

17.  MODIFICATIONS OF THE METHOD
Rats treated with reserpine develop spontaneous
orofacial dyskinesia that has features similar to
tardive dyskinesia in humans (Nisewander et al.
1994).
 The incidence of tongue protrusions was recorded to
quantify the occurrence of oral dyskinesia.

18. d. Circling behavior in nigrostriatal
lesioned rats
PURPOSE AND RATIONALE
Unilateral lesion of the dopaminergic nigrostriatal
pathway in the rat by the neurotoxin 6-
hydroxydopamine (6OHDA) induces hypersensitivity
of the postsynaptic dopaminergic receptors in the
striatum of the lesioned side.
The rats rotate in a direction towards the lesioned
side (ipsilateral) when an indirect acting compound
such
as amphetamine is administered but to the opposite
direction (contralateral) when a direct acting
dopamine agonist, e.g., apomorphine, or the
dopamine precursor L-dopa is given.

19. Therefore, this test can be used for the study of
central dopamine function and the evaluation of
dopamine antagonists and agonists, particularly the
activity of novel antiparkinsonian drugs.
 MODIFICATIONS OF THE METHOD
Etemadzadeh et al. (1989) described a computerized
rotometer apparatus for recording circling behavior
Hudson et al. (1993) described a 16-channel
automated rotometer system for reliable
measurement of turning behavior in 6-
hydroxydopamine lesioned and transplanted rats
Garrett and Holtzman (1996) compared the effects of
apomorphine, cocaine and caffeine on locomotor
activity and rotational behavior in rats with unilateral
6-OHDA-induced lesions of the nigrostriatal tract.

20. e. Elevated body swing test
 PURPOSE AND RATIONALE
Borlongan and Sanberg (1995) proposed the elevated
body swing test as a measure of asymmetrical motor
behavior of hemiparkinsonian animals in a drug-free
state.
f. Skilled paw reaching in rats
 PURPOSE AND RATIONALE
Barnéoud et al. (1996) used the skilled paw reaching
test as a model of Parkinson’s disease in the rat.

21. Unilateral injection of 6-OHDA into the medial
forebrain bundle results in an impairment of paw
reaching on both sides which can be ameliorated by
drug treatment or transplantation of a nigral cell
suspension.
g. Stepping test in rats
 PURPOSE AND RATIONALE
Schallert et al. (1992), Olsson et al. (1995) introduced
the stepping test as a clinically relevant unilateral
model of Parkinsonian akinesia.
The 6-OHDA lesion induced marked and long-lasting
impairments in the initiation of stepping movements
with the contralateral paw which can be ameliorated
by the systemic application of drugs.

22. II. IN VITRO AND EX VIVO MODELS
a.Dopamine- stimulated Adenylyl cyclase activity.

23. REFERANCES
Drug Discovery and Evalution by H.
Gerhard Vogel. II nd Ed.
Drugs Screening Methods By S. K.
Gupta.
www.google.com

24. Thank You