Lecture No: MPL103_19

Lecture title: Screening of Anti-depressants

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 Lecture Objectives
By the end of this Lecture, students will be able to:
Discuss various experimental methods available to evaluate
antidepressant drugs using different models

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 Contents
Screening of Anti-depressants

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 DEPRESSION:
• Depression is defined as a “mental state of altered mood
characterized by feelings of Sadness, Loss of interest, Disturbed
sleep, Disturbed appetite, Low energy, Poor concentration,
Hopelessness.
PRINCIPLE:-
Monoamine theory-1965
Depression is caused by the functional deficit of monoamine
transmitters at certain sites of brain.
Mania results from a functional excess of these transmitters.

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 SCREENING METHODS
IN VITRO METHODS IN VIVO METHODS
i. Inhibition of [3H]- nor I. Behavioral tests
epinephrine uptake in a) Despair swim test.
rat brain synaptosomes b) Tail suspension test in mice.
ii. Inhibition of [3H] - c) Muricide behavior in rats.
dopamine uptake in rat II. Test for antidepressant activity based on the
striatal synaptosome. MAO.
iii. Inhibition of [3H] - a) Potentiation of nor epinephrine toxicity
serotonin uptake in b) Apomorphine- induced hypothermia in mice
synaptosome. c) Reserpine induced hypothermia.

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 INHIBITION OF NOREPINEPHRINE UPTAKE IN RAT BRAIN
PRINCIPLE:-
Reuptake of norepinephrine is inhibited by antidepressant drugs. This mode
of action leads to receptor down regulation.
PROCEDURE:-
TISSUE PREPARATION:
• Male wistar rats are decapitated and the brains removed rapidly.
Hypothalamic region is prepared, weighed, and homogenised in 9 volumes
ice cold 0.32 M sucrose solution using a potter-Elevejhem homogeniser .
• Homogenate is centrifuged at 1000g at 0-4ºc for 10 min. The supernatant
decanted and used for the uptake expt.

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 ASSAY:-

• 200µl of tissue suspension + 800ml NE krebs – henseleit

bicarbonate buffer +20µl of drug concentration are incubated at
37ºc under 95% O2 / 5% CO2 atmosphere for 5min.
• For each assay, 3 tubes are incubated with 20µl of vehicle at Oºc
in an ice bath.
• After incubation all tubes are centrifuged at 4000g for 10min.
• The supernatant fluid is aspirated and the pellets dissolved in 1ml
of solubilizer (tritonX-100+ 50% ethanal 1:4) .
• The tubes are shaken , decanted into scintillation vials, counted in
10ml of liquid Scintillation .
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 • Add radioactive ligand for that receptor in presence and
absence of test drugs count the receptor ligand binding by
liquid scintillography.
• Active uptake is the difference b/w counts per minute at 37ºc
and Ooc
EVALUATION:-
The percent inhibition at each drug concentration is determined
and compare with standard

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 INVIVO METHODS
DESPAIR SWIM TEST:-
PRINCIPLE:-
Mice/rats forced to swim in restricted space from which they
cannot escape
immobility

This behavior reflects a state of despair reduced by several agents

which are therapeutically effective in human depression.

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 PROCEDURE:

• Male Sprague- Dawley rats weighing 160-180g are used.

• Rats are individually forced to swim inside vertical Plexiglas cylinder
(height: 40cm; diameter: 18cm, containing 15cm of water maintained
at 25° c)
• Rats placed in the cylinders for first times are initially highly active,
vigorously swimming in circles, trying to climb to wall or diving to the
bottom.
• After 2-3 min activity begin to subside and immobility starts.
• An animal is judged to be immobile whenever it remain floating
passively in the water in a slightly hunched but up right position, its
nose just above the surface.
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 • After 15 min in the water the rats are removed and allowed to
dry in a heated enclosure (32°c) before being returned to their
home cage.
• They are again placed in the cylinder 24hr later and the total
duration of immobility is measured during a 5min test.
• Test drug or standard are administered one hour prior the
testing.

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 EVALUATION:-
Duration of immobility is measured in controls and animals
treated with various dose of a test drug or standard.

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 TAIL SUSPENSION TEST IN MICE

PRINCIPLE:-
• The tail suspension test has been described by Steru et al. for evaluating
potential antidepressants.
• The immobility displayed by rodents when subjected to an avoidable and
inescapable stress .
• Clinically effective antidepressants reduce the immobility that mice display
after active and unsuccessful attempts to escape when suspended by the tail.

PROCEDURE:-
• Male Balb mice weighting 20-25g are used preferentially. They are housed in
plastic cage for at least 10 days prior to testing in a 12hr light cycle with food
and water freely available.

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 • Group of 10 animals are treated with the test compounds or the
vehicle by intraperitoneal injection 30min prior to testing.
• For the test the mice are suspended on the edge of a shelf 58cm
above a table top by adhesive tape placed approximately 1cm from
the tip of the tail.
• The duration of immobility is recorded for a period of 5 min. mice
are considered immobile when hang passively and completely
motionless for at least 1min.

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 EVALUATION:
•The percentage of animals showing the passive behavior is
counted and compared with vehicle treated controls.

• Using various doses, ED50 values can be calculated.

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 TESTS FOR ANTIDEPRESSANT ACTIVITY BASED ON THE
MECHANISM OF ACTION
POTENTIATION OF NOREPINEPHRINE TOXICITY:-
PRINCIPLE:-
Antidepressants block the re-uptake of biogenic amines into nervous
tissue. In this way, the toxic effects of norepinephrine are potentiated.
PROCEDURE:-
• Male NMRI mice (22-25g) are randomly assigned to test groups of 10
subjects.
• The test drug, the standard or the vehicles are given orally1 hr prior to
the s.c. injection of the sub lethal dose of 3mg/kg noradrenaline.
• The groups of 10 mice are placed into plastic cages with free access to
food and water. 16
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 EVALUATION:
The mortality rate is assessed 48hr post-dosing. ED 50 or dose
which causes death of 50% of the treated subjects is calculated by
means of linear regression analysis.

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 APOMORPHINE- INDUCED HYPOTHERMIA IN MICE
PRINCIPLE:-
Apomorphine induces hypothermia in mice which can be prevented by
antidepressants .
PROCEDURE:
• Male NMRI mice (20-22g) are used and randomly assigned to test groups of 6
subjects.
• One hour after oral administration of the test compounds or the vehicle a dose
of 16mg/kg apomorphine is injected s.c.
• The rectal temperature of each mouse is measured by an electronic
thermometer immediately prior to apomorphine administering and 10. 20 and
30 min later.
• During the entire experiment, subjects are housed in groups of three jars at
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 EVALUATION:
A time curve is constructed by plotting the temperature against
time in min. the AUC is calculated for all groups and converted
into percent inhibition of Apomorphine- induced hypothermia in
the control group. An ED50 can calculate by linear regression
analysis.

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 9. Rota Rod Test

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 Summary
This lecture dealt with the preclinical screening methods like

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