Models for screening the test compound. Through this models, the evaluation of test drug can be done.

1. CNS STIMULANTS &
DEPRESSANTS SCREENING MODELS
By: DHRUVI PATEL
Sub: Pharmacological and
Toxicological Screening Methods
[MPL103T]

2. WHAT ARE CNS STIMULANTS?
▪Central nervous system stimulants may be used to reduce
tiredness and increase alertness, competitiveness and
aggression.
▪CNS stimulants may be defined as “Drug substance that
most specifically afford an enhancement in excitability
either very much within the different portions of the brain
or the spinal cord which may lead to convulsion.”

3. BEHAVIOURAL MANIFESTATION OF CNS
STIMULATION
▪ Analeptic effect- Alertness and Attention
Nervousness and Anxiety
Drowsiness and Fatigue
▪ Can also lead to convulsions

4. CLASSIFICATION OF CNS STIMULANTS
1. Analeptics (convulsant)
• Doxapram (respiratory stimulation)
• Nikethamide (respiratory stimulant)
• Strychnine
• Picrotoxin
• Bicuculline
2. Psychomotor stimulants
• Amphetamine & Methamphetamine
• Cocaine
• Methylphenidate
• Phenetamine
• Fenfluramine

5. 3. Methylxanthines derivatives
• Theophylline (tea)
• Caffeine (coffee)
• Theobromine (chocolate)
4. MAO Inhibitors
• Phenelzines
• Tranylcypromine
5. Catecholamines reuptake inhibitors

6. EVALUATION METHODS
In-vivo methods-
1. Screening of analeptics by Actophotometer
2. Runway test
3. Open Field test
4. Hole Board test
5. Combined Open Field test
6. Strychnine induced convulsion
7. Ptosis test
8. Sand displacement method

7. SCREENING BY ACTOPHOTOMETER
▪ Purpose: To evaluate locomotor activity in animal as most of the drugs acting on
the CNS, influences the locomotor activity on the animal.
▪ Principle: When the beam of light falling on the photocell is cut off by the animal,
it is recorded in the terms of count by digital counter.
▪ Procedure: Weigh animals (mice 30-40g) and label them. Now measure the
normal (control) reading in actophotometer and denote the count with basal
activity score.
▪ After injection of stimulant drug, record the movement of mice in the
actophotometer after 30mins.
▪ Evaluation: Increase in locomotor activity score
indicate the CNS stimulant property of the drug.

8. RUNWAY TEST
▪ Principle: TheY-shaped Runway is covered with paper that can indicate the
footprints of mice which is counted afterwards for evaluation.
▪ Procedure: Wistar rats of either sex weighing 250-300gm are grouped.
The control group is placed in the centre of the Y and leaving it in the apparatus for
5mins.The number of times it enter the Arms is recorded as a measure of activity in
the normal condition.
After 30mins of administration of test drug, the same procedure is been followed.
Now the number of time it enters the arm is recorded.To measure the degree of
ataxia, the floor of apparatus is covered by a clean paper.
▪ Evaluation:
The number of foot prints on the maze part is measured.
Higher number indicates the drug has stimulant effect.

9. OPEN FIELD TEST
▪ Purpose: To test general levels of locomotion activity and anxiety in animal.
▪ Principle: Interruption of light beams as a measure of movements of rats or mice
in an Open field.
▪ Procedure: The rats (280-320gm) are placed and observed in square open field
that has 25 boxes drawn and arena (100x100x40cm) equipped with photocells,
sensitive to infrared light.
Now after 15mins of subcutaneous injection of test drug, observe the rat for 5mins in
the open field apparatus.
The number of boxes crossed and interruptions of photo beams in the lower rows
shows the reading for motor activity.
The interruptions of photo beams in the upper rows shows reading for rearing.
▪ Evaluation: Higher the reading of motor activity and rearing as compared to
control group indicates the drug is CNS stimulant.

10. Open field apparatus:

11. HOLE BOARD TEST
▪ Purpose: The evaluation of behaviour of mice such as curiosity or exploration.
▪ Principle: An open field with holes on the bottom is used into which the animals
could poke their noses which is a sign of curiosity.
▪ Procedure: The hole board has a size of 40x40cm with 16 holes having diameter
of 3cm each are distributed evenly on floor.
Mice of either sex (20-25gm) are placed on the board and nose poking is measured
by visual observation. Nose poking is thought to indicate curiosity.
Now measure the behaviour 30mins after the administration of the test compound for
5mins.
▪ Evaluation: The number of counts for nose poking of treated animals is compared
to that of control animals.
Higher the nose poking count indicates higher curiosity, which means the drug
shows CNS stimulant effect.

12. Hole board apparatus-

13. COMBINED OPEN FIELD TEST
▪ Purpose: The simultaneous determination of locomotion and curiosity.
▪ Principle: By a modification of the hole board and a photobeam system.
▪ Procedure: Mice with an average weight of 30gm are used.
In addition a row of 4 photocell beams is mounted 1cm outside of the holes which
automatically records every exploratory nose poke.
30mins after i.p and 60mins after oral administration of the test compound the animal
is placed into the cage and behaviour is recorded for a period of 5mins.
▪ Evaluation: Counts for motility (interruption of photocell beam inside the cage)
and for curiosity (interruption of photocell beam outside the cage due to nose
poking) are recorded and compared with control.
Higher interruption indicates drug to be CNS stimulant.

14. STRYCHNINE INDUCED CONVULSION
▪ Purpose: To evaluate the convulsive effect.
▪ Principle: The convulsing action of strychnine is due to interference with
postsynaptic inhibition mediated by glycine. Strychnine-sensitive postsynaptic
inhibition in higher centres of the CNS is also mediated by glycine. Compounds
which reverse the action of strychnine have been shown to have anxiolytic
properties.
▪ Procedure: Group of 10 mice of either sex with average weight between 20-25gm
are used.
They are treated orally with test compound. One hour later the mice are injected
with strychnine nitrate i.p. The time until occurrence of tonic extensor convulsion
and death is noted during 1hr period.
▪ Evaluation: Lesser time required for occurrence of tonic extensor convulsion and
death more potent convulsive CNS stimulant the drug is.

15. PTOSIS TEST
▪ Purpose: To evaluate the CNS stimulant/depressant activity.
▪ Principle: Reserpine causes the complete ptosis (depression like state) owing to
central depression & this state is useful in evaluating CNS stimulant.
▪ Procedure: Albino mice of either sex weighing 20-25gm are used.
Reserpine is given i.p (4mg/kg) to the animal. Complete ptosis is reached at about
3 hours.
After 2hr 45min of reserpine injection the test drug administered. After 15mins
ptotic rating is made. 4 for complete ptosis, 2 for half ptosis and 0 for normal eyelid.
▪ Evaluation: If the ptosis rating is observed to be 0, it means the test drug is potent
CNS stimulant.

16. A- Normal eyelid
B- Ptosis condition

17. SAND DISPLACEMENT METHOD
▪ Purpose: To evaluate the motor activity in animals.
▪ Principle: Drug like caffeine stimulate the motor activity in animal.
▪ Procedure: A cage with sand is calibrated for amount of sand displaced per unit
time.
Then mice weighing 25-30gm are divided in 3 groups.
Control- saline, standard- Caffeine(20mg/kg), test- drug to be evaluated.
After 30mins the animal is placed in cage for 15mins.
▪ Evaluation: Volume of sand displaced in each group is compared.
More sand displaced- CNS stimulant Activity.
Less sand displaced- CNS depressant Activity.

18. WHAT ARE CNS DEPRESSANT?
▪ CNS depressants are type of drugs that slows down brain activity, which causes the
muscles to relax, calm and soothes a person making them useful for treating
anxiety, panic, acute stress reaction and sleep disorders.
EMOTIONAL SYMPTOMS-
• A loss of pleasure and interest in usual activities and work.
• Anxiety symptoms are present in 90% patients.
• Low self esteem and loss of motivation.
• Retardation of thoughts and actions.
PHYSICAL SYMPTOMS-
• Chronic fatigue with decreased ability to perform normal daily tasks.
• Poor muscular incoordination and reduced locomotion.
• Difficulty in falling asleep.

19. CLASSIFICATION OF CNS DEPRESSANT
1. Sedatives
Barbiturates such as-
• Phenobarbital
• Pentobarbital sodium
2. Hypnotics
Non-benzodiazepines such as-
• Zolpidem
• Zaleplon
3. Tranquilizer
Benzodiazepines such as-
• Diazepam
• Clonazepam
• Triazolam

20. EVALUATION METHODS
In-vivo methods-
1. Despair swim test
2. Tail suspension test
3. Rotarod test
4. Actophotometer
5. Tread wheel test
6. Chimney test
7. Test for motor coordination
• Horizontal bar
• Static rod
• Hanging wire

21. In-vitro methods-
1. Inhibition of [3H]-norepinephrine uptake in rat brain synaptosomes
2. Inhibition of[3H]-dopamine uptake in rat striatal synaptosomes
3. Inhibition of [3H]-serotonin uptake in synaptosomes
4. Antagonism of p-chloramphetamine toxicity by inhibitors of serotonin uptake
5. Receptor subsensitivity after treatment with antidepressant:
Simultaneous determination of the effect of chronic antidepressant treatment on
beta-adrenergic and 5-HT2 receptor densities in rat cerebral cortex.
6. Measurement of beta-adrenoreceptor stimulated adenylate cyclase
7. [3H]-Yohimbine binding to alpha2 adrenoreceptors in rat cerebral cortex
8. Test for anticholinergic properties by [3H]-QNB binding to muscarinic
cholinergic receptors in rat brain
9. Monoamine oxidase inhibition: Inhibition of type A and type B monoamine
oxidase activity in rat brain synaptosomes

22. DESPAIR SWIM TEST
▪ Purpose: To check behavioural related activity.
▪ Principle: Mice are placed in an inescapable tank and forced to swim in a
restricted space hence their escape related motility behaviour is measured.
▪ Procedure: Rat weighing 200-250gm are used.
Apparatus setup- a vertical plexiglass cylinder (height 50cm; diameter 18cm,
containing 30cm of water maintained at 25 degree)
Rat used placed in the cylinders for the first time are initially highly active,
vigorously swimming in circles, trying to climb the wall or diving to the bottom. After
2-3mins the activity begins to subside. After 5-6min rat remain immobile for 80% of
time.
After 1 hour of administration of test compound the animal is placed in the cylinder
to check the behavioural changes.
▪ Evaluation: Duration of immobility is measured in control and animal treated with
test drug.

23. More the immobile duration of time spent by rat indicates that the test drug is Potent
CNS depressant.
If the animal is struggling/climbing in the cylinder then the drug has moderate CNS
depressant activity.
Swimming of animal implies that the drug has no CNS depressant activity.

24. TAIL SUSPENSION TEST
▪ Purpose: This model is a modification of behaviour despair test.
▪ Principle: Behavioural test useful in the screening of potential antidepressant
drugs.
▪ Procedure: Tail suspension boxes made of plastic with the dimension (55height;
60width; 12cm depth) are used.
Mice weighting 20-25gm are selected and used for the test.
After 30mins of i.p administration of test compound, the mice is suspended on the
top of suspension box with the help of adhesive tape.The tape is placed
approximately 1cm away from the tip of the tail.
The duration of immobility is observed and recorded for the period of 5mins.
▪ Evaluation: The time that each animal spends as mobile with comparison to that of
control animal group is measured.
Higher the immobility time in test animal group, implies the test compound is CNS
depressant.

25. Tail suspension test

26. ROTAROD TEST
▪ Purpose: To evaluate the effect of drug on motor coordination.
▪ Principle: The length of the time that a given animal stays on the rotating rod is a
measure of their balance, coordination and physical condition.
▪ Procedure: Mice weighing 20-30gm are used.
After the test compound is administered i.p (30mins) or orally (1hour), the mice are
placed on the rotating rod.
The speed of apparatus is adjusted to 2 rotation per minute.
The time of animal demonstrating their ability to remain on the revolving rod is
recorded (ideally for 1min).
▪ Evaluation: The time for falling of animal is recorded.
Less time indicates the drug to be CNS depressant.

27. Rotarod apparatus

28. TREAD WHEEL TEST
▪ Purpose: To evaluate effect of drug on locomotor activity of animal.
▪ Principle: Based on the number of rotation on tread wheel on which the animal
moves.
▪ Procedure: Wistar rats of either sex weighing 250-300gm are used.
Animal placed in tread wheel for 10mins to observe number of rotations.
After 30mins of i.p administration of test compound, the animal is placed on the tread
wheel and the number of rotations are recorded for 10mins.
Compare the number of rotations of control and that of standard group.
▪ Evaluation:
Higher number of rotations implies the drug to be CNS stimulant
And vice versa.

29. CHIMNEY TEST
▪ Purpose: To evaluate muscle coordination and locomotor activity of animal.
▪ Principle: The animal reacts spontaneously and performs coordinated movement
similar to an alpinist to pass a chimney in the mountains.
▪ Procedure: Male mice weighing 20-25gm are used.
30cm long Pyrex glass cylinder is used. Initially the cylinder is held in a horizontal
position, at the end of cylinder near a 20cm, mark from the base. A mice is
introduced with the head forward.When it reaches the mark on the other end of the
cylinder, the tube is moved to a vertical position. Immediately the mouse tries to
climb backward & performs coordinated movement.The time required by the mouse
to climb backwards to the top of cylinder is noted.
After the test dose administration, repeat the procedure and record the time.
Compare the time for control and test group.
▪ Evaluation: More the time required by the mouse to climb backward, indicates
the drug to be CNS depressant.

30. TEST FOR MUSCLE COORDINATION
▪ Purpose: To evaluate the motor coordination or disfunction of muscles.
▪ Principle: Motor incoordination is detected by failure of mice to hold on the bar,
rod and wire for longer period of time.
The first manifestation of depression of central nervous system in the mouse is motor
weakness and incoordination.

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