Screening models on behavioral and muscle coordination converted

SCREENING MODELS on BEHAVIORAL
AND
MUSCLE COORDINATION
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Department of Pharmacology BVVS COP BGK

❖ BENZODIAZEPINES
➢ Benzodiazepines act preferentially on mid brain ascending reticular formation (which
maintains wakefulness) & on limbic system (thought & mental function).
➢ BZDs act by enhancing presynaptic/postsynaptic inhibition through a specific BZD
receptor which is an integral part of GABA a receptor- Cl channel complex.
➢ Only α and β subunits are required for GABA action and most likely the binding site for
GABA is located on beta subunit.
➢ While alpha or gama subunits interface carries the BZD receptor increases the frequency
of chlorine channel opening induced by submaximal concentration of GABA.
➢ BZD and newer α-1 selective hypnotics (e.g. zolpidem) bind to a site located between
α and γ subunits, which facilitates channel opening when binding occurs.
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➢ BZDs produce centrally mediated skeletal muscle relaxation without impairing voluntary
activity.
➢ Clonazepam and diazepam have more marked muscle relaxant property.
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a) Motor activity and behavior:
1)Method of intermittent observation
2)Open field test
3)Hole board test
4)Combined open field test
b) Muscle coordination:
1)Inclined plane method
2)Chimney test
3)Grip strength
4)Rota rod method
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a) Motor activity and behavior:
1. Method of intermittent observation
Purpose:
➢Study stimulant and sedative drugs by changing animal behavior after
administration of drug.
Procedure:
➢ Mice weight 25g, 12 animals are divided into 4 groups of 3 animals.
➢ One group of 3 mice as control & other groups receive different doses of test
drug intraperitoneal.
➢ For testing stimulant activity, the mice are injected after 10 min s.c with
800mg/kg methamphetamine.
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➢ For testing sedative activity, the mice are treated with 0.5mg/kg paraldehyde.
➢ Each group is placed into a glass jar of 12cm diameter and 20cm height
which in turn are placed in a wooden box of 130 X 50.30cm.
➢ The glass jars are illuminated from above.
➢ 10min after administration of methamphetamine or paraldehyde observation
is started.
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❖ EVALUATION:
➢ During 1hr the observer looks every minute for 1s to each jar and registers if one, two or
all three or none mice show any characteristic change in locomotion, rearing, grooming or
sniffing.
➢ The maximum count in 1hr would be 180.
➢ Generally, activity decreases within 1 hr.
➢ Nevertheless, methamphetamine treated animals have a total count between 120 and 150.
➢ Groups treated with an effective sedative drug shows a dose dependent decrease of total
counts.
➢ The numbers of counts in the treated groups is calculated as % of controls.
➢ From dose response curves ED50 values can be calculated.
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2. Open field test:
Purpose:
➢ To assess the activity of sedative or stimulant of drugs.
Procedure:
➢ The rats are observed in square open field area(68X68X45cm) equipped with
2 rows of 8 photocells, sensitive to infrared light, placed 40 and 125mm
above the floor.
➢ Measurements are made in the dark ventilated sound attenuating box.
➢ Interruption of photocell beam can be collected by a microcomputer
and Interruption of light beams as a movements of rats or mice in a
cage.
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❖ EVALUATION:
➢ Dose response curve can be obtained for sedative and stimulant drugs, whereby the various
parameters show different results.
➢ The effects of various doses are compared statistically with the values of controls and
among themselves.
❖ MODIFICATIONS OF THE METHOD:
➢ Vorhees et al. (1992) described a locomotor activity system for rodents.
➢ The system consists of black, ventilated test chamber, internally lighted with a
ceiling mounted video camera.
➢ The camera’s image is transmitted to a contrast and relays the digitalized
coordinates to a PC.
➢ Dedicated software stores the information and simultaneous displays a map of the
tracked subject.
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3. HOLE-BOARD TEST:
❖Purpose and Rational:
➢The evaluation of certain components of behavior of mice such as curiosity or exploration
has been attempted by Boissier et al.(1964) and Boissier and Simon (1964).
➢They used an open field with holes on the bottom into which the animals could poke their
noses.
❖PROCEDURE:
➢ Mice of either sex with a weight between 18 and 22g are used.
➢ The hole-board has a size of 40X40cm. Sixteen holes with a diameter of 3cm each are
distributed evenly on the floor.
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➢ The board is elevated so that the mouse poking its nose into the hole does not
see the bottom.
➢ Nose poking is thought to indicate curiosity and is measured by visual
observation in the earliest description and counted by electronic devices in
more recent modifications.
➢ Usually, 6 animals after administration of the test compound the first animal
is placed on the hole board and tested for 5 min.
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❖EVALUATION:
➢The number of counts for nose poking of treated animals is calculated as
percentage of control animals.
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b) Test for muscle coordination:
1) Chimney test:
Purpose:
A simple test for assessing the tranquilizing and muscle relaxant activity.
Procedure:
➢ Male mice weighing between 16-22gm are used.
➢ Pyrex glass cylinders of 30cm long are required.
➢ The internal diameter varies with the animals weight For mice 16-18gm, the
diameter is (22mm), 18-20gm (25mm), 20-22gm (28mm).
➢ Each tube has a mark 20cm from its base. Initially, the tube is held in a
horizontal position. At the end of the tube, near the mark, a mouse is
introduced at with the head forward.
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➢ When the mouse reaches the other end of the tube, toward which it is pushed
with a rod if necessary with a rod the tube is moved to a vertical position.
➢ Immediately the mouse tries to climb backwards and performs coordinated
movements.
➢ The time required by the mouse to climb backwards out at the top of the
cylinder is noted.
❖ EVALUATION:
➢ The ED50, the dose for which 50% of the animals fail to climb backwards
out of the tube within 30s.
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2. Grip strength :
❖Purpose:
➢ This test is used to assess muscular strength or neuromuscular function.
❖Procedure:
➢ Male or female mice with an average. Weight of 22gm are used.
➢ The animals are exposed to a horizontal thin threat metallic wire suspended
about 30cm into the air which they immediately grasp with the forepaws.
➢ The mouse is released to hang on with its forelimbs. Normal animals are able
to catch the threat with the hind limbs and to climb up within 5sec.
➢ After oral or s.c administration. The animals are tested every 15 min animals
which are not able to touch the threat with the hind limbs within the 5sec or
fail off from the threat are considered to be impaired.
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❖EVALUATION:
➢ The percentage of animals loosing the catching reflex is calculated.
➢ By use of different doses, ED50 values are calculated.
➢ Likewise, time response curves can be established.
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❖MODIFICATIONS OF THE TEST:
➢ Simianet et al.(1989) used a test originally described by Fleury (1957).
➢ A mouse held by the tail is placed on a small metallic grid which the animal gripped with
its forepaws.
➢ The grid is than loaded with weights until the mouse could not longer support the weight
of the grid.
➢ The end point is the maximal weight supported by the animal at least for 2s.
➢ ED50 values can be calculated for various centrally active skeletal muscle relaxants such
as the benzodiazepines.
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3. ROTA ROD METHOD:
Purpose:
➢ The test is used to evaluate the activity of drugs interfering with motor co-
ordination and muscle activity.
Procedure:
➢ Male mice with an weight between 20-30gm are used.
➢ The apparatus consists of a horizontal wooden rod coated with rubber with
3cm diameter. Attached to a motor with the speed adjusted to a 2rpm .
➢ The rod is 75cm in length and is divided into 6 sections by plastic discs,
there by allowing the simultaneous testing of 6 mice. The rod is in height of
about 50cm above the table top in order to discourage the animals from
jumping off the roller.
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➢ Cages below the section serve to restrict the movements of the animals when they fall from
the roller.
➢ Only those animals which have demonstrated their ability to remain on the revolving rod
for at least 1 min are used for the test.
➢ The test compound is administrated by i.p or orally.
➢ 30 min after i.p or 60 min after oral administration. The mice are placed for 1min on the
rotating rod.
➢ The number of animals falling from the roller during this time is counted.
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ROTAROD METHOD
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❖ EVALUATION:
➢ Percentage animals falling from the rota rod within the test period is calculated for every
drug concentration tested.
➢ ED50 is defined as the dose of drug at which 50% of the test animals fall from the rota
rod.
❖ MODIFICATIONS OF THE TEST:
➢ Rozas et al.(1997) described a drug free rota rod test that was used to evaluate the effects
of unilateral 6-hydroxydopamine lesions, nigral grafts, and sub rotational doses of
apomorphine.
➢ The rota rod unit was automated and interfaced with a personal computer allowing
automatic recording of the time that each rat was able to stay on the rod at different
rotational speeds.
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❖ REFERENCES:
➢ Rang H.P., Dale M.M., Ritter J.M., Flower. R.J.Rang and Dale’s
Pharmacology.
➢ K.D Tripathi 7th edition.
➢ Drug Discovery & Evaluation Pharmacological assay – H. G. Vogel
➢ Goodman and Gilman`s Manual of Pharmacology and Therapeutics
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THANK YOU
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Screening models on behavioral and muscle coordination converted

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Screening models on behavioral and muscle coordination converted